α-Terpinyl Acetate: Occurrence in Essential Oils Bearing Thymus pulegioides, Phytotoxicity, and Antimicrobial Effects.

The aim of this study was to evaluate occurrence of T. pulegioides α-terpinyl acetate chemotype, as source of natural origin α-terpinyl acetate, to determine its phytotoxic and antimicrobial features. Were investigated 131 T. pulegioides habitats. Essential oils were isolated by hydrodistillation and analyzed by GC-FID and GC-MS. Phytotoxic effect of essential oil of this chemotype on monocotyledons and dicotyledons through water and air was carried out in laboratory conditions; the broth microdilution method was used to screen essential oil effect against human pathogenic microorganisms. Results showed that α-terpinyl acetate was very rare compound in essential oil of T. pulegioides: it was found only in 35% of investigated T. pulegioides habitats. α-Terpinyl acetate (in essential oil and pure) demonstrated different behavior on investigated plants. Phytotoxic effect of α-terpinyl acetate was stronger on investigated monocotyledons than on dicotyledons. α-Terpinyl acetate essential oil inhibited seeds germination and radicles growth for high economic productivity forage grass monocotyledon Poa pratensis, but stimulated seed germination for high economic productive forage legume dicotyledon Trifolium pretense. α-Terpinyl acetate essential oil showed high antimicrobial effect against fungi and dermatophytes but lower effect against bacteria and Candida yeasts. Therefore, T. pulegioides α-terpinyl acetate chemotype could be a potential compound for developing preventive measures or/and drugs for mycosis.

Toxic, Radical Scavenging, and Antifungal Activity of Rhododendron tomentosum H. Essential Oils.

The chemical composition of eight (seven shoot and one inflorescence) essential oils (EOs) of Rh. tomentosum H. plants growing in Eastern Lithuania is reported. The plant material was collected during different phases of vegetation (from April to October). The oils were obtained by hydrodistillation from air-dried aerial parts (leaves and inflorescences). In total, up to 70 compounds were identified by GC-MS and GC (flame-ionization detector, FID); they comprised 91.0 ± 4.7%-96.2 ± 3.1% of the oil content. Sesquiterpene hydrocarbons (54.1 ± 1.5%-76.1 ± 4.5%) were found to be the main fraction. The major compounds were palustrol (24.6 ± 2.6%-33.5 ± 4.4%) and ledol (18.0 ± 2.9%-29.0 ± 5.0%). Ascaridol isomers (7.0 ± 2.4%-14.0 ± 2.4% in three oils), myrcene (7.2 ± 0.3% and 10.1 ± 1.3%), lepalol (3.3 ± 0.3% and 7.9 ± 3.0%), and cyclocolorenone isomers (4.1 ± 2.5%) were determined as the third main constituents. The toxic activity of marsh rosemary inflorescence and shoot oils samples was evaluated using a brine shrimp (Artemia sp.) bioassay. LC50 average values (11.23-20.50 µg/mL) obtained after 24 h of exposure revealed that the oils were notably toxic. The oil obtained from shoots gathered in September during the seed-ripening stage and containing appreciable amounts of palustrol (26.0 ± 2.5%), ledol (21.5 ± 4.0%), and ascaridol (7.0 ± 2.4%) showed the highest toxic activity. Radical scavenging activity of Rh. tomentosum EOs depended on the plant vegetation stage. The highest activities were obtained for EOs isolated from young shoots collected in June (48.19 ± 0.1 and 19.89 ± 0.3 mmol/L TROLOX (6-hydroxy-2,5,7,8-tetra-methylchromane-2-carboxylic acid) equivalent obtained by, respectively, ABTS•+ (2,2'-amino-bis(ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and DPPH•(2,2-diphenyl-1-picrylhydrazyl) assays). Agar disc diffusion assay against pathogenic yeast Candida parapsilosis revealed the potential antifungal activity of EOs. An alternative investigation of antifungal activity employed mediated amperometry at yeast Saccharomyces cerevisiae-modified electrodes. The subjection of yeast cells to vapors of EO resulted in a three to four-fold increase of electrode responses due to the disruption of yeast cell membranes.

Initiation of radioecological monitoring of forest soils and plants at the Lithuanian border region before the start of the Belarusian nuclear power plant operation.

Knowledge of the background activity concentrations of anthropogenic radionuclides before the start of operation of the new nuclear power plant in Belarus, BelNPP, is an issue of great importance for neighbouring countries. In this study, we provide the pilot characterisation of the Lithuanian part of the 30-km zone of the BelNPP, emphasising the forest plants, terrestrial mosses, forest organic and mineral topsoil to describe the preoperational radioecological state of the pine forest ecosystem. Key anthropogenic radionuclides (14C, 3H, 137Cs and 239,240Pu) were analysed. The 14C specific activity varied from 97.80 ± 1.30 to 102.40 ± 0.79 pMC. The 3H specific activity in the tissue-free water tritium form varied from 13.2 ± 2.2 TU to 20.8 ± 2.3 TU, which corresponded to the 3H level of precipitation in this region. The activity concentrations of 239,240Pu in soil and moss samples did not exceed 1 Bq/kg and were mainly due to global fallout after nuclear tests. The 137Cs inventory in the pine forest soils of the Lithuanian part of the BelNPP 30-km zone varied from 930 ± 70 to 1650 ± 430 Bq/m2. High variation of the inventory and uneven distribution in the soil profile conditioned a wide range of 137Сs activity in terrestrial plants from 1.0 ± 0.5 to 40.5 ± 1.8 Bq/kg dry weight. The abundance of microorganisms in different seasons and soil depths do not exceed the natural levels. According to PCA loads, the number of microorganisms and variability of 137Cs specific activity is determined by soil abiotic parameters.

Low concentrations of acetic and formic acids enhance the inactivation of Staphylococcus aureus and Pseudomonas aeruginosa with pulsed electric fields.

BACKGROUND: Skin infections, particularly caused by drug-resistant pathogens, represent a clinical challenge due to being a frequent cause of morbidity and mortality. The objectives of this study were to examine if low concentrations of acetic and formic acids can increase sensitivity of Staphylococcus aureus and Pseudomonas aeruginosa to pulsed electric field (PEF) and thus, promote a fast and efficient treatment methodology for wound treatment. RESULTS: We have shown that the combination of PEF (10-30 kV/cm) with organic acids (0.1% formic and acetic acids) increased the bactericidal properties of treatment. The effect was apparent for both acids. The proposed methodology allowed to reduce the energy of electrical pulses and the inhibitory concentrations of acids, while still maintain high efficiency of bacteria eradication. CONCLUSIONS: Application of weak organic acids as bactericidal agents has many advantages over antibiotics because they do not trigger development of drug-resistance in bacteria. The combination with PEF can make the treatment effective even against biofilms. The results of this study are particularly useful for the development of new methodologies for the treatment of extreme cases of wound infections when the chemical treatment is no longer effective or hinders wound healing.

Membrane Permeabilization of Pathogenic Yeast in Alternating Sub-microsecond Electromagnetic Fields in Combination with Conventional Electroporation.

Recently, a novel contactless treatment method based on high-power pulsed electromagnetic fields (PEMF) was proposed, which results in cell membrane permeabilization effects similar to electroporation. In this work, a new PEMF generator based on multi-stage Marx circuit topology, which is capable of delivering 3.3 T, 0.19 kV/cm sub-microsecond pulses was used to permeabilize pathogenic yeast Candida albicans separately and in combination with conventional square wave electroporation (8-17 kV/cm, 100 µs). Bursts of 10, 25, and 50 PEMF pulses were used. The yeast permeabilization rate was evaluated using flow cytometric analysis and propidium iodide (PI) assay. A statistically significant (P < 0.05) combinatorial effect of electroporation and PEMF treatment was detected. Also the PEMF treatment (3.3 T, 50 pulses) resulted in up to 21% loss of yeast viability, and a dose-dependent additive effect with pulsed electric field was observed. As expected, increase of the dB/dt and subsequently the induced electric field amplitude resulted in a detectable effect solely by PEMF, which was not achievable before for yeasts in vitro.

Induction of Different Sensitization Patterns of MRSA to Antibiotics Using Electroporation.

Treatment of bacteria-associated infections is complicated and antibiotic treatment alone is often inadequate to overcome biofilm infections. Physical methods allow overcoming this problem and propose solutions that are non-dependent on drug resistance. In this work, we investigated the feasibility of pulsed electric fields for sensitization of MRSA to common antibiotics. We analyzed the efficacy of inactivation of methicillin-resistant Staphylococcus aureus in 5⁻20 kV/cm electric field separately and in combination with gentamicin, doxycycline, ciprofloxacin, sulfamethoxazole, and vancomycin. Combined treatment allowed using up to 1000-fold smaller concentrations of antibiotics to induce the same inactivation of S. aureus.

Antifungal properties of hypericin, hypericin tetrasulphonic acid and fagopyrin on pathogenic fungi and spoilage yeasts.

CONTEXT: The role of hypericin-mediated photodynamic antimicrobial properties on pathogenic fungi and photodynamic therapy for human cancer cells is known. Antifungal properties of Hypericum perforatum L. (Hypericaceae) and Fagopyrum esculentum Moench. (Polygonaceae) extracts were also studied. The different polarities of solvents can cause complication in the identification of antifungal effects of separate biologically active compounds. In recent experimental work, we compared antifungal properties of purified hypericin, hypericin tetrasulphonic acid (hypericin + S) and fagopyrin, which is analogue of hypericin. OBJECTIVE: The antifungal properties of aromatic polyketide derivatives such as hypericin, hypericin + S and fagopyrin on the selected pathogenic fungi and spoilage yeasts have been studied. MATERIALS AND METHODS: The antifungal properties of hypericin, hypericin + S and fagopyrin were determined using the broth microdilution method against a set of pathogenic fungi and spoilage yeasts including: Microsporum canis, Trichophyton rubrum, Fusarium oxysporum, Exophiala dermatitidis, Candida albicans, Kluyveromyces marxianus, Pichia fermentans and Saccharomyces cerevisiae. The tested concentrations of hypericin, hypericin + S and fagopyrin ranged from 750 to 0.011 µg/mL and MIC values were evaluated after 48 h incubation at 30 °C. RESULTS: The results confirm different antifungal properties of hypericin, hypericin + S and fagopyrin on the selected pathogenic fungi and spoilage yeasts. For pathogenic fungi, the minimum inhibitory concentrations of hypericin ranged 0.18-46.9 µg/mL, hypericin + S 0.18-750 µg/mL and fagopyrin 11.7-46.9 µg/mL. For spoilage yeasts, the MICs of hypericin and hypericin + S ranged 0.18-46.9 and 0.011-0.73 µg/mL, respectively. DISCUSSION AND CONCLUSION: The results obtained herein indicate that various chemical structures of hypericin, hypericin + S and fagopyrin can develop different antifungal properties.

Irreversible electropermeabilization of the human pathogen Candida albicans: an in-vitro experimental study.

Pathogenic fungi cause many life-threatening infections, especially among individuals with immune system dysfunction. The antifungal drugs commonly used to suppress fungal pathogens can result in long-lasting and toxic therapy. In this work, irreversible electropermeabilization was used to investigate the dynamics of the decrease in Candida albicans colony vitality after application of a pulsed electric field (PEF) and use of antifungal drugs. The fungi were subjected to single 250-µs to 2-ms (0.5-2.5 kV/cm) pulses or repeated short 5-µs pulses, and efficacy was compared. It was shown that electropermeabilization combined with antifungal agents results in rapid and more effective treatment, eliminating more than 90% of C. albicans colony-forming units in a single procedure, which is advantageous in biomedicine. It was also observed that, because of application of PEF and use of the antifungal agents, the Candida cells form cell aggregates and average live cell size is reduced by as much as 53%.

Succession of microorganisms in a plate-type air treatment biofilter during filtration of various volatile compounds.

Changes in the number and species diversity of cultivable microorganisms in a newly developed plate-type biofilter during filtration of various volatile pollutants were studied. The novelty of the investigation is the monitoring of microorganism succession in different parts of biofilter plates with original packing material consisting of birch fibre and needle-punched non-woven fabric. It was shown that the largest number of fungi and yeasts develop on the top and middle, while bacteria develop on the bottom and middle parts of plates. The number of microorganisms depends on the origin of the pollutant, the pH and temperature inside the biofilter and the moisture of the porous plates. The statistically significant correlation between the number of microorganisms and inlet concentration of acetone was estimated, while ammonia showed a negative influence on yeast distribution. Paecilomyces variotii, Rhodotorula mucilaginosa and Bacillus subtilis were the most common organisms found during filtration of all examined volatiles; however, some differences of microbial communities in different parts of the biofilter plates and filtrated volatile compounds were obtained.

Microsecond pulsed magnetic field improves efficacy of antifungal agents on pathogenic microorganisms.

Control and treatment of the emerging filamentous and yeast fungal diseases are of high priority in the biomedical field. This study investigated the influence of the pulsed magnetic field combined with common antifungal agents on the viability of various pathogenic fungi such as Aspergillus fumigatus, Candida albicans, and Trychophyton rubrum. Repetitive microsecond pulsed magnetic fields up to 6.1 T were applied in the study. The synergistic effect of co-applying drugs and magnetic treatment to different fungi species causing various human mycoses showed the potential for more effective and less toxic therapy.

First Data on the Investigation of Gut Yeasts in Hermit Beetle (Osmoderma barnabita Motschulsky, 1845) Larvae in Lithuania.

In this study, yeasts from the gut of O. barnabita larvae were isolated and molecularly identified. It is worth noting that this research provides the first analysis of the gut yeast community in O. barnabita larvae in Lithuania, which is a significant contribution to the field. Two hermit-like L3-praepupa instars were collected from a decaying oak log in Lithuania. The isolation, morphology, biochemistry, and physiology of the yeast isolates were characterized using standards commonly employed in yeast taxonomy studies. The isolates were identified by sequencing the large subunit (26S) rDNA (D1/D2 domain of the LSU). All gut compartments were colonized by the yeast. A total of 45 yeast strains were obtained from the gut of both O. barnabita larvae, with 23 strains originating from Larva 1, 16 strains from Larva 2, and 6 strains from the galleries. According to our identification results of the 45 yeast strains, most of the species were related to Ascomycota, with most of them belonging to the Saccharomycetales order. Yeasts of the genera Candida, Debaryomyces, Meyerozyma, Priceomyces, Schwanniomyces, Spencermartinsiella, Trichomonascus, and Blastobotrys were present in gut of O. barnabita larvae. Species of the Trichosporonales order represented the Basidiomycota phylum.

Impact of Soil Chemical Properties on the Growth Promotion Ability of Trichoderma ghanense, T. tomentosum and Their Complex on Rye in Different Land-Use Systems.

Microbial-based biostimulants that increase plant performance and ensure sustainable restoration of degraded soils are of great importance. The aim of the present study was to evaluate the growth promotion ability of indigenous Trichoderma ghanense, T. tomentosum and their complex on early rye seedlings in sustained grassland and arable soil. The impact of soil chemical properties on the ability of selected Trichoderma strains and their complex to promote plant growth was determined by the evaluation of the rye (Secale cereale L.) early seedling growth-measuring the length of shoots and roots as well as their dry weight. Trichoderma species were tested for their ability to produce extracellular degradative enzymes on solid media. Furthermore, the soil properties and CM-cellulase activity of soil were estimated. The indigenous Trichoderma strains possess the capacity to produce enzymes such as peroxidase, laccase, tyrosinase, and endoglucanase. The results indicated a significant (p < 0.05) increase in plant growth and the improvement of some soil chemical properties (total N, mobile humic and fulvic acids, exchangeable K2O, soil CM-cellulase activity) in inoculated soils when compared to the control. The growth of the roots of rye seedlings in sustained grassland was enhanced when T. tomentosum was applied (p = 0.005). There was an increase in total weight and shoot weight of rye seedlings when T. ghanense was used in the arable soil (p = 0.014 and p = 0.024). The expected beneficial effect of Trichoderma spp. complex on rye growth promotion was not observed in any tested soil. The results could find application in the development of new and efficient biostimulants, since not only do physiological characteristics of fungi play an important role but also the quality of the soil has an impact.

In vitro inhibition of Saccharomyces cerevisiae growth by Metschnikowia spp. triggered by fast removal of iron via two ways.

Simple and convenient innovative assays in vitro demonstrating Metschnikowia spp. competition with Saccharomyces cerevisiae for an essential nutrient iron are presented. The tested Metschnikowia strains possess a common genetically determined property of secreting a pulcherriminic acid which in the presence of iron (III) ions forms an insoluble red pigment pulcherrimin. Both initial accumulation in growing Metschnikowia cells and subsequent precipitation in the form of pulcherrimin in the media contribute to iron removal by functioning cells. The predominant way depends on the strain. Due to fast elimination of iron, the growth of S. cerevisiae can be inhibited by tested Metschnikowia strains at concentrations of elemental iron in the media not exceeding 12 mg kg-1. Inhibition can be regulated by additional supply of microquantities of iron onto the surface of the solid medium within 20-24 h. At relatively low concentrations of elemental iron (below 1 mg kg-1), additional supplements of iron onto the surface provide an advancement in understanding the inhibition possibilities and enable the assay control. Microscopy observations revealed that Metschnikowia chlamydospores are involved in iron removal at relatively high iron concentrations. The results may find application in development of new methodologies and strategies for biocontrol or inhibition of pathogenic microorganisms.

Candida Distribution in Onychomycosis and in vitro Susceptibility to Antifungal Agents.

The aim of this study was to determine distribution of the Candida (C.) species in onychomycosis and analyses in vitro susceptibility to fluconazole and itraconazole. In recent years, cases of onychomycosis in Lithuania caused by Candida have increased significantly. In the period between 2009 and 2016, a total of 8149 clinical cases (outpatients and inpatients) were investigated at the Vilnius University Hospital Santaros Clinics (VUH SC). Candida yeasts were identified using VITEK 2 (BioMerieux, France) and IVD Maldi biotyper 2.3 (Bruker Daltonik GmbH, Germany), automated systems for identification of yeasts. The antifungal susceptibility to the Candida species were determined by disc diffusion. Candida spp. were the most frequently isolated pathogens in onychomycosis during the investigation period. The main species in onychomycosis were C. albicans (38.6%), followed by C. krusei (33.7%), C. tropicalis (11.1%), C. parapsilosis (7.9%), and other Candida (8.7%). The different antifungal susceptibility patterns among Candida species confirm the need to perform antifungal susceptibility in vitro testing of yeasts from patients with onychomycosis.

An application of advanced hair-save processes in leather industry as the reason of formation of keratinous waste: few peculiarities of its utilisation.

The application of more environmentally friendly hide and skin unhairing technologies in leather processing results in a significant increase in keratin waste. There are currently two most promising hair-saving unhairing methods: enzymatic and hair immunisation. The complete use of hair-saving unhairing methods in the leather industry will lead to the formation of approximately 143 thousand tons of hair/wool waste annually, which will require disposal. The disposal of keratin wastes from the leather industry has not been adequately studied, bearing in mind the possible amount of such wastes that will be produced in the future. Unfortunately, existing studies pay little attention to the method of unhairing, even though the unhairing method has a vast influence on the properties of keratin in the obtained hair/wool wastes. Accordingly, the present research is an attempt to establish how the differently obtained keratin wastes behave following disposal. The obtained results have shown that waste wool is characterised by different behaviour during burial in soil, and the behaviour depends on the method of unhairing. This proposition is valid for waste wool bioresistance as well. It was concluded that the deterioration of any sort of keratinous waste from the leather industry should be investigated thoroughly before disposal by burial in landfills.

Nanosecond duration pulsed electric field together with formic acid triggers caspase-dependent apoptosis in pathogenic yeasts.

Antifungal substances that are used for the treatment of candidiasis have considerable side effects and Candida yeasts are known to obtain drug resistance. The multidrug resistance cases are promoting the search for the new alternative methods and pulsed electric field (PEF) treatment could be the alternative or could be used in combination with conventional therapy for the enhancement of the effect. We have shown that nanosecond range PEF is capable to induce apoptosis in the S. cerevisiae as well as in the drug resistant C. lusitaniae and C. guilliermondii. Supplementing the PEF procedure with formic acid (final concentration 0.05%) resulted in improvement of the inactivation efficacy and the induction of apoptosis in the majority of the yeast population. After the treatment yeast were displaying the DNA strand brakes, activation of yeast metacaspase and externalization of phosphatidylserine. Apoptotic phenotypes were registered already after 30 kV/cm × 250 ns × 50 pulses treatment. The highest number of apoptotic yeast cells (>60%) was obtained during the 30 kV/cm × 750 ns × 50 pulses protocol when combined with 0.05% formic acid. The results of our study are useful for development of new non-toxic and effective protocols to induce programed cell death in different yeast species and thus minimize inflammation of the tissue.

Influence of plant origin natural α-pinene with different enantiomeric composition on bacteria, yeasts and fungi.

Although the nature-identical chemical compounds are cheaper, they not always repeat biological activity of plant origin natural chemical compounds, often react allergies and resistance of microorganisms. The aim of this study was to investigate effects of Juniperus communis origin α-pinene with different enantiomeric composition on bacteria, yeasts and fungi. Results showed that different enantiomeric composition of α-pinene have different activities on microorganisms: essential oil with (1S)-(-) ≈ (1R)-(+) enantiomeric composition of α-pinene influenced on some microorganisms stronger than essential oil with (1S)-(-) < (1R)-(+) enantiomeric composition of α-pinene; the pure natural α-pinene with enantiomeric composition S < R more strongly inhibited growth of investigated bacteria and Candida yeasts, α-pinene with enantiomeric composition S ≈ R - growth of Trichophyton and Aspergillus. (1S)-(-) and (1R)-(+) enantiomeric forms of α-pinene can have also different synergistic effects with other compounds of essential oil. The results of study showed that the same amount of α-pinene with different enantiomeric composition can have diverse antimicrobial potential due different specific interactions with other chemical compounds of essential oil. Therefore, it is very important to determine and present the enantiomeric composition of those plant origin compounds, which are characterized by enantiomerisation, during the course of research of biological activities of natural plant products (essential oils and other) and their isolated compounds.

Pulsed electric field-assisted sensitization of multidrug-resistant Candida albicans to antifungal drugs.

AIM: Determine the influence of pH on the inactivation efficiency of Candida albicans in pulsed electric fields (PEF) and evaluate the possibilities for sensitization of a drug-resistant strain to antifungal drugs. MATERIALS & METHODS: The effects of PEF (2.5-25 kVcm-1) with fluconazole, terbinafine and naftifine were analyzed at a pH range of 3.0-9.0. Membrane permeabilization was determined by flow cytometry and propidium iodide. RESULTS: PEF induced higher inactivation of C. albicans at low pH and increased sensitivity to terbinafine and naftifine to which the strain was initially resistant. Up to 5 log reduction in cell survival was achieved. CONCLUSION: A proof of concept that electroporation can be used to sensitize drug-resistant microorganisms was presented, which is promising for treating biofilm-associated infections.

Different permeabilization patterns of splenocytes and thymocytes to combination of pulsed electric and magnetic field treatments.

Genetic manipulation of T cells is frequently inefficient, however, when combined with physical methods (i.e. electroporation) a promising alliance with immunotherapy can be formed. This study presents new data on permeabilization of murine thymocytes and splenocytes as a T cell model using pulsed electric (PEF) and electromagnetic field (EMF). The 300ns, 500ns, 2µs and 100µs pulse bursts in a broad range of PEF 0-8kV/cm were applied separately and in combination with 3.3T, 0.2kV/cm EMF pulses. The permeabilization efficiency was evaluated using fluorescent dye (YO-PRO-1) and flow cytometry. It was shown that a >14% increase in thymocytes permeabilization is achieved when electroporation is applied in combination with EMF, however splenocytes responded in a different manner - a statistically significant (P<0.05) reduction in permeabilization was observed. The cytokine secretion patterns were mainly unaltered independently on the applied treatment parameters determined by secretion of IFNγ, IL-4 and IL-17 - the main cytokines of Th1, Th2 and Th17 cells. The results of this study are useful for development of pulsed power protocols for effective genetic modification of T cells.

Non-invasive nanosecond electroporation for biocontrol of surface infections: an in vivo study.

Invasive infections caused by drug-resistant bacteria are frequently responsible for fatal sepsis, morbidity and mortality rates. In this work, we propose a new methodology based on nanosecond high frequency electric field bursts, which enables successful eradication of bacteria in vivo. High frequency (15 kHz) 15-25 kV/cm 300-900 ns pulsing bursts were used separately and in combination with acetic acid (0.1-1%) to treat Pseudomonas aeruginosa in a murine model. Acetic acid 1% alone was effective resulting in almost 10-fold reduction of bacteria viability, however combination of nanosecond electric field and acetic acid 1% treatment was the most successful showing almost full eradication (0.01% survival compared to control) of the bacteria in the contaminated area. The short duration of the pulses (sub-microsecond) and high frequency (kHz range) of the burst enabled reduction of the muscle contractions to barely detectable level while the proposed applicators ensured predominantly topical treatment, without electroporation of deeper tissues. The results of our study have direct application for treatment of wounds and ulcers when chemical treatment is no longer effective.