Inactivation of microorganisms by plasma of a positive (PC) and negative corona (NC) discharge in air at atmospheric pressure was investigated. Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Pseudomonas aeruginosa were chosen for the discharge inactivation. PC and NC produce three types of bactericidal agents, which are ultraviolet radiation (UV), neutral reactive species (R), and electric field and charged particles (E), respectively. We elucidated the contribution of each bioactive agent to the inactivation of S. aureus and P. aeruginosa. The influence of the charged particles in PC and NC on the cell inactivation is caused by different electrophysical effects, which lead nevertheless to an identical consequence: the cell membrane becomes more transparent for neutral reactive species. It gives additional possibility for neutral reactive species to increase the inactivation of cell by biochemical mechanisms. Due to that, total UV þ R þ E bactericidal effect in PC and NC is approximately the same and great -only a few tens of seconds is enough to inactivate completely S. aureus and P. aeruginosa cells.

Chronic infections are associated with the formation of non-attached biofilm-like aggregates. In vitro models of surface-attached biofilms do not always accurately mimic these processes. Here, we tested a new approach to create in vitro non-attached bacterial aggregates using the principle of magnetic levitation of biological objects placed into a magnetic field gradient. Bacteria grown under magnetic levitation conditions formed non-attached aggregates that were studied with CLSM and SEM and characterized quantitatively. Non-attached aggregates consisted of bacteria submerged into an extracellular matrix and demonstrated features characteristic of biofilms, such as polymeric matrix that binds Ruby Red and Congo red dyes, prerequisite of bacterial growth, and increased resistance to gentamicin. Three quantitative methods were explored to characterize strain-specific potential to form non-attached aggregates: geometric sizes, relative quantities of aggregated and free-swimming bacteria, and Congo red binding. A comparison of three E. coli strains demonstrated that the strain weakly forming non-attached aggregates differed from strains that formed aggregates based on all three parameters (p<0.05). Further, we characterized biofilm formation on plastic and agar surfaces by these strains and found that good biofilm formation ability does not necessarily indicate good non-attached aggregate formation ability, and vice versa. The model and quantitative methods can be applied for in vitro studies of non-attached aggregates and modeling bacterial behavior in chronic infections, as it is important to increase understanding of the role that non-attached bacterial aggregates play in the pathogenesis of chronic diseases. Importance paragraph An increasing amount of evidence indicates that chronic infections are associated with non-attached biofilm-like aggregates formed by pathogenic bacteria. These aggregates differ from biofilms because they form under low-shear conditions within the volume of biological fluids and they do not attach to surfaces. Here, we describe an in vitro model that provides non-attached aggregate formation within the liquid volume due to magnetic levitation. Using this model, we demonstrated that despite morphological and functional similarities of non-attached aggregates and biofilms, strains that exhibit good biofilm formation might exhibit poor non-attached aggregate formation, suggesting that mechanisms underlying the formation of biofilms and non-attached aggregates are not identical. The magnetic levitation approach can be useful for in vitro studies of non-attached aggregate formation and simulation of bacterial behavior in chronic infections.

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Non-thermal plasma (NTP) is a flow of partially ionized argon gas at an ambient macroscopic temperature and is microbicidal for bacteria, viruses and fungi. Viability of the Gram-negative obligate intracellular bacterial parasite Chlamydia trachomatis and its host cells was investigated after NTP treatment. NTP treatment of C. -fold reduction in the concentration of infectious bacteria. When the samples were covered with magnesium fluoride glass to obstruct plasma particles and UV rays alone were applied, the bactericidal effect was reduced 1.4¾10 1 -fold and 5¾10 4-fold for EBs and RBs, respectively. NTP treatment caused the viability of host McCoy cells to diminish by 19 %. Therefore, the results obtained demonstrated that (i) both extracellular and intracellular forms of C. trachomatis are sensitive to NTP treatment; (ii) the reduction in concentration of infectious bacteria after NTP treatment of infected cells is superior to the reduction in viability of host cells; and (iii) the effect of NTP on intracellular bacteria does not depend on UV rays.

Aim: To study the effects exerted by argon microwave nonthermal plasma (NTP) on cell wall-lacking Mollicutes bacteria. Methods and Results: 10 8 CFU ml À1 agar plated Mycoplasma hominis and Acholeplasma laidlawii were treated with the nonthermal microwave argon plasma for 30-300 s. The maximal 10-and 100-fold drop was observed for A. laidlawii and Myc. hominis, respectively. Similarly treated Escherichia coli and Staphylococcus aureus demonstrated the 10 5 and 10 3 drop, respectively. Removal of cholesterol affected resistance of A. laidlawii. 10 mmol l À1 antioxidant butylated hydroxytoluene decreased mortality by a factor of 25-200. UV radiation alone caused 25-85% mortality in comparison with the whole NTP. Exogenously added hydrogen peroxide H 2 O 2 did not cause mortality. NTP treatment of Myc. hominis triggered growth of microcolonies, which were several tenfold smaller than a typical colony. Conclusions: Despite the lack of cell wall, A. laidlawii and Myc. hominis were more resistant to argon microwave NTP than other tested bacteria. Mycoplasma hominis formed microcolonies upon NTP treatment. A role of UV and active species was demonstrated. Significance and Impact of the Study: The first study of NTP effects on Mollicutes revealed importance of a membrane composition for bacterial resistance to NTP. New specific Myc. hominis morphological forms were observed. The study confirmed importance of the concerted action of reactive oxygen species (ROS) with UV and other plasma bioactive agents for NTP bactericidal action.