Overcoming antibiotic resistance: non-thermal plasma and antibiotics combination inhibits important pathogens.

Antibiotic resistance (ATBR) is increasing every year as the overuse of antibiotics (ATBs) and the lack of newly emerging antimicrobial agents lead to an efficient pathogen escape from ATBs action. This trend is alarming and the World Health Organization warned in 2021 that ATBR could become the leading cause of death worldwide by 2050. The development of novel ATBs is not fast enough considering the situation, and alternative strategies are therefore urgently required. One such alternative may be the use of non-thermal plasma (NTP), a well-established antimicrobial agent actively used in a growing number of medical fields. Despite its efficiency, NTP alone is not always sufficient to completely eliminate pathogens. However, NTP combined with ATBs is more potent and evidence has been emerging over the last few years proving this is a robust and highly effective strategy to fight resistant pathogens. This minireview summarizes experimental research addressing the potential of the NTP-ATBs combination, particularly for inhibiting planktonic and biofilm growth and treating infections in mouse models caused by methicillin-resistant Staphylococcus aureus or Pseudomonas aeruginosa. The published studies highlight this combination as a promising solution to emerging ATBR, and further research is therefore highly desirable.

Modeling the growth of Aspergillus brasiliensis affected by a nonthermal plasma.

AIM: The main objective of the study was to develop and validate a model for the growth of Aspergillus brasiliensis on surfaces, specifically on agar culture medium. An additional aim was to determine conditions for complete growth inhibition of this micromycete using two different nonthermal plasma (NTP) sources. METHODS AND RESULTS: The developed model uses two key parameters, namely the growth rate and growth delay, which depend on the cultivation temperature and the amount of inoculum. These parameters well describe the growth of A. brasiliensis and the effect of NTP on it. For complete fungus inactivation, a single 10-minute exposure to a diffuse coplanar surface barrier discharge was sufficient, while a point-to-ring corona discharge required several repeated 10-minute exposures at 24-h intervals. CONCLUSIONS: The article presents a model for simulating the surface growth of A. brasiliensis and evaluates the effectiveness of two NTP sources in deactivating fungi on agar media.

Comparison of the Effect of Plasma-Activated Water and Artificially Prepared Plasma-Activated Water on Wheat Grain Properties.

Recently, much attention has been paid to the use of low-temperature plasmas and plasma-activated water (PAW) in various areas of biological research. In addition to its use in medicine, especially for low-temperature disinfection and sterilization, a number of works using plasma in various fields of agriculture have already appeared. While direct plasma action involves the effects of many highly reactive species with short lifetimes, the use of PAW involves the action of only long-lived particles. A number of articles have shown that the main stable components of PAW are H2O2, O3, HNO2, and HNO3. If so, then it would be faster and much more practical to artificially prepare PAW by directly mixing these chemicals in a given ratio. In this article, we review the literature describing the composition and properties of PAW prepared by various methods. We also draw attention to an otherwise rather neglected fact, that there are no significant differences between the action of PAW and artificially prepared PAW. The effect of PAW on the properties of wheat grains (Triticum aestivum L.) was determined. PAW exposure increased germination, shoot length, and fresh and dry shoot weight. The root length and R/S length, i.e., the ratio between the underground (R) and aboveground (S) length of the wheat seedlings, slightly decreased, while the other parameters changed only irregularly or not at all. Grains artificially inoculated with Escherichia coli were significantly decontaminated after only one hour of exposure to PAW, while Saccharomyces cerevisiae decontamination required soaking for 24 h. The differences between the PAW prepared by plasma treatment and the PAW prepared by artificially mixing the active ingredients, i.e., nitric acid and hydrogen peroxide, proved to be inconsistent and statistically insignificant. Therefore, it may be sufficient for further research to focus only on the effects of artificial PAW.

A Review of Microbial Decontamination of Cereals by Non-Thermal Plasma.

Cereals, an important food for humans and animals, may carry microbial contamination undesirable to the consumer or to the next generation of plants. Currently, non-thermal plasma (NTP) is often considered a new and safe microbicidal agent without or with very low adverse side effects. NTP is a partially or fully ionized gas at room temperature, typically generated by various electric discharges and rich in reactive particles. This review summarizes the effects of NTP on various types of cereals and products. NTP has undisputed beneficial effects with high potential for future practical use in decontamination and disinfection.

Non-thermal Plasma Treatment of ESKAPE Pathogens: A Review.

The acronym ESKAPE refers to a group of bacteria consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. They are important in human medicine as pathogens that show increasing resistance to commonly used antibiotics; thus, the search for new effective bactericidal agents is still topical. One of the possible alternatives is the use of non-thermal plasma (NTP), a partially ionized gas with the energy stored particularly in the free electrons, which has antimicrobial and anti-biofilm effects. Its mechanism of action includes the formation of pores in the bacterial membranes; therefore, resistance toward it is not developed. This paper focuses on the current overview of literature describing the use of NTP as a new promising tool against ESKAPE bacteria, both in planktonic and biofilm forms. Thus, it points to the fact that NTP treatment can be used for the decontamination of different types of liquids, medical materials, and devices or even surfaces used in various industries. In summary, the use of diverse experimental setups leads to very different efficiencies in inactivation. However, Gram-positive bacteria appear less susceptible compared to Gram-negative ones, in general.

Inactivation of Dermatophytes Causing Onychomycosis Using Non-Thermal Plasma as a Prerequisite for Therapy.

Following our previous study of the therapy of onychomycosis by non-thermal plasma (NTP) and nail hygiene and to obtain some prerequisite data of dermatophytes sensitivity, the dynamics of those inactivation by NTP plasma was monitored for various strains of Trichophyton iterdigitale, Trichophyton benhamiae, Trichophyton rubrum, and Microsporum canis. Three strains of each species on agar plates were exposed with plasma produced by a DC corona discharge in the point-to-ring arrangement in various time intervals. Although all strains were sufficiently sensitive to plasma action, significant differences were observed in their sensitivity and inactivation dynamics. These differences did not correlate with the species classification of individual strains, but could be assigned to four arbitrarily created types of strain response to NTP according to their sensitivity. These results indicate that the sensitivity to plasma is not an inherent property of the fungal species, but varies from strain to strain.

Inactivation of Acanthamoeba Cysts in Suspension and on Contaminated Contact Lenses Using Non-Thermal Plasma.

Water suspensions of cysts of a pathogenic clinical isolate of Acanthamoeba sp. were prepared, and the cysts were inactivated either in suspension or placed on the surface of contact lenses by the non-thermal plasma produced by the DC corona transient spark discharge. The efficacy of this treatment was determined by cultivation and the presence of vegetative trophozoites indicating non-inactivated cysts. The negative discharge appeared to be more effective than the positive one. The complete inactivation occurred in water suspension after 40 min and on contaminated lenses after 50 min of plasma exposure. The properties of lenses seem to not be affected by plasma exposure; that is, their optical power, diameter, curvature, water content and infrared and Raman spectra remain unchanged.

Effects of Non-Thermal Plasma Treatment on Seed Germination and Early Growth of Leguminous Plants-A Review.

The legumes (Fabaceae family) are the second most important agricultural crop, both in terms of harvested area and total production. They are an important source of vegetable proteins and oils for human consumption. Non-thermal plasma (NTP) treatment is a new and effective method in surface microbial inactivation and seed stimulation useable in the agricultural and food industries. This review summarizes current information about characteristics of legume seeds and adult plants after NTP treatment in relation to the seed germination and seedling initial growth, surface microbial decontamination, seed wettability and metabolic activity in different plant growth stages. The information about 19 plant species in relation to the NTP treatment is summarized. Some important plant species as soybean (Glycine max), bean (Phaseolus vulgaris), mung bean (Vigna radiata), black gram (V. mungo), pea (Pisum sativum), lentil (Lens culinaris), peanut (Arachis hypogaea), alfalfa (Medicago sativa), and chickpea (Cicer aruetinum) are discussed. Likevise, some less common plant species i.g. blue lupine (Lupinus angustifolius), Egyptian clover (Trifolium alexandrinum), fenugreek (Trigonella foenum-graecum), and mimosa (Mimosa pudica, M. caesalpiniafolia) are mentioned too. Possible promising trends in the use of plasma as a seed pre-packaging technique, a reduction in phytotoxic diseases transmitted by seeds and the effect on reducing dormancy of hard seeds are also pointed out.

Non-Thermal Plasma Sources Based on Cometary and Point-to-Ring Discharges.

A non-thermal plasma (NTP) is a promising tool against the development of bacterial, viral, and fungal diseases. The recently revealed development of microbial resistance to traditional drugs has increased interest in the use of NTPs. We have studied and compared the physical and microbicidal properties of two types of NTP sources based on a cometary discharge in the point-to-point electrode configuration and a corona discharge in the point-to-ring electrode configuration. The electrical and emission properties of both discharges are reported. The microbicidal effect of NTP sources was tested on three strains of the bacterium Staphylococcus aureus (including the methicillin-resistant strain), the bacterium Pseudomonas aeruginosa, the yeast Candida albicans, and the micromycete Trichophyton interdigitale. In general, the cometary discharge is a less stable source of NTP and mostly forms smaller but more rapidly emerging inhibition zones on agar plates. Due to the point-to-ring electrode configuration, the second type of discharge has higher stability and provides larger affected but often not completely inhibited zones. However, after 60 min of exposure, the NTP sources based on the cometary and point-to-ring discharges showed a similar microbicidal effect for bacteria and an individual effect for microscopic fungi.

Inactivation of Schistosoma Using Low-Temperature Plasma.

The inactivation of Schistosoma mansoni cercariae and miracidia was achieved by exposure to plasma produced by the positive, negative, and axial negative corona discharges. The positive discharge appeared as the most effective, causing the death of cercariae and miracidia within 2-3 min of exposure. The negative discharge was less effective, and the axial discharge was ineffective. The water pre-activated (PAW) by the discharges showed similar efficiency, with the exception of the significantly effective PAW activated with axial discharge. These facts, together with the observation of various reactions among plasma-damaged schistosomes, suggest that the mechanisms of inactivation by different types of discharges are different.

Polylactic acid as a suitable material for 3D printing of protective masks in times of COVID-19 pandemic.

A critical lack of personal protective equipment has occurred during the COVID-19 pandemic. Polylactic acid (PLA), a polyester made from renewable natural resources, can be exploited for 3D printing of protective face masks using the Fused Deposition Modelling technique. Since the possible high porosity of this material raised questions regarding its suitability for protection against viruses, we have investigated its microstructure using scanning electron microscopy and aerosol generator and photometer certified as the test system according to the standards EN 143 and EN 149. Moreover, the efficiency of decontaminating PLA surfaces by conventional chemical disinfectants including 96% ethanol, 70% isopropanol, and a commercial disinfectant containing 0.85% sodium hypochlorite has been determined. We confirmed that the structure of PLA protective masks is compact and can be considered a sufficient barrier protection against particles of a size corresponding to microorganisms including viruses. Complete decontamination of PLA surfaces from externally applied Staphylococcus epidermidis, Escherichia coli, Candida albicans and SARS-CoV-2 was achieved using all disinfectants tested, and human adenovirus was completely inactivated by sodium hypochlorite-containing disinfectant. Natural contamination of PLA masks worn by test persons was decontaminated easily and efficiently by ethanol. No disinfectant caused major changes to the PLA surface properties, and the pore size did not change despite severe mechanical damage of the surface. Therefore, PLA may be regarded as a suitable material for 3D printing of protective masks during the current or future pandemic crises.

Inactivation of Dermatophytes Causing Onychomycosis and Its Therapy Using Non-Thermal Plasma.

Onychomycosis is one of the most common nail disorders. Its current treatment is not satisfactorily effective and often causes adverse side effects. This study aims to determine the optimal conditions for non-thermal plasma (NTP) inactivation of the most common dermatophytes in vitro and to apply it in patient`s therapy. The in vitro exposure to NTP produced by negative DC corona discharge caused full inactivation of Trichophyton spp. if applied during the early growth phases. This effect decreased to negligible inactivation with the exposure applied six days after inoculation. In a group of 40 patients with onychomycosis, NTP therapy was combined with nail plate abrasion and refreshment (NPAR) or treatment with antimycotics. The cohort included 17 patients treated with NPAR combined with NTP, 11 patients treated with antimycotics and NTP, and 12 patients treated with NPAR alone. The combination of NPAR and NTP resulted in clinical cure in more than 70% of patients. The synergistic effect of NPAR and NTP caused 85.7% improvement of mycological cure confirmed by negative microscopy and culture of the affected nail plate. We conclude that NTP can significantly improve the treatment of onychomycosis.

Use of non-thermal plasma pre-treatment to enhance antibiotic action against mature Pseudomonas aeruginosa biofilms.

Non-thermal plasma (NTP), generated at atmospheric pressure by DC cometary discharge with a metallic grid, and antibiotics (gentamicin-GTM, ceftazidime-CFZ and polymyxin B-PMB), either alone or in combination, were used to eradicate the mature biofilm of Pseudomonas aeruginosa formed on Ti-6Al-4V alloy. Our aim was to find the conditions for NTP pre-treatment capable of enhancing the action of the antibiotics and thus reducing their effective concentrations. The NTP treatment increased the efficacy of relatively low concentrations of antibiotics. Generally, the highest effect was achieved with GTM, which was able to suppress the metabolic activity of pre-formed P. aeruginosa biofilms in the concentration range of 4-9 mg/L by up to 99%. In addition, an apparent decrease of biofilm-covered area was confirmed after combined NTP treatment and GTM action by SYTO®13 staining using fluorescence microscopy. Scanning electron microscopy confirmed a complete eradication of P. aeruginosa ATCC 15442 mature biofilm from Ti-6Al-4V alloy when using 0.25 h NTP treatment and subsequent treatment by 8.5 mg/L GTM. Therefore, NTP may be used as a suitable antibiofilm agent in combination with antibiotics for the treatment of biofilm-associated infections caused by this pathogen.

Combination of non-thermal plasma and subsequent antibiotic treatment for biofilm re-development prevention.

The influence of non-thermal plasma (NTP) treatment on the prevention of antibiotic resistance of microbial biofilms was studied. Staphylococcus epidermidis and Escherichia coli bacteria and a yeast Candida albicans, grown on the surface of Ti-6Al-4V alloy used in the manufacture of prosthetic implants, were employed. Their biofilms were exposed to NTP produced by DC cometary discharge and subsequently treated with antibiotics commonly used for the treatment of infections caused by them: erythromycin (ERY), polymyxin B (PMB), or amphotericin B (AMB), respectively. All biofilms displayed significant reduction of their metabolic activity after NTP exposure, the most sensitive was S. epidermidis. The subsequent action of antibiotics caused significant decrease in the metabolic activity of S. epidermidis and E. coli, but not C. albicans, although the area covered by biofilm decreased in all cases. The combined effect of NTP with antibiotics was thus proved to be a promising strategy in bacterial pathogen treatment.

Medically important biofilms and non-thermal plasma.

In recent decades, the non-thermal plasma, i.e. partially or completely ionized gas produced by electric discharges at ambient temperature, has become of interest for its microbiocidal properties with potential of use in the food industry or medicine. Recently, this interest focuses not only on the planktonic forms of microorganisms but also on their biofilms. The works in this interdisciplinary field are summarized in this review. The wide range of biofilm-plasma interactions is divided into studies of general plasma action on bacteria, on biofilm and on its oral and dental application; a short overview of plasma instrumentation is also included. In addition, not only biofilm combating but also an important area of biofilm prevention is discussed. Various DC discharges of the point-to-plane type. Author's photograph, published in Khun et al. (Plasma Sources Sci Technol 27:065002, 2018).

Inactivation of dermatophyte infection by nonthermal plasma on animal model.

Superficial fungal infections are a major epidemiological issue with increasing prevalence and are a common global problem. This article describes experimental therapy of superficial fungal skin infection using low-temperature plasma. Groups of guinea pigs were artificially infected with Trichophyton mentagrophytes SK 3286 dermatophyte and treated with plasma produced by a DC cometary discharge with an inserted grid. The course of infection was a week shorter and milder in animals treated by plasma than that in nontreated animals, the significant lowering of dermatophytic germs also occurred in the treated group. The exposure to plasma causes no harm to experimental animals. The results allow for the development of a new dermatophytoses therapy by low temperature plasma treatment.

Inactivation of human pathogenic dermatophytes by non-thermal plasma.

Non-thermal plasma (NTP) was tested as an in vitro deactivation method on four human pathogenic dermatophytes belonging to all ecological groups including anthropophilic Trichophyton rubrum and Trichophyton interdigitale, zoophilic Arthroderma benhamiae, and geophilic Microsporum gypseum. The identification of all strains was confirmed by sequencing of ITS rDNA region (internal transcribed spacer region of ribosomal DNA). Dermatophyte spores were suspended in water or inoculated on agar plates and exposed to NTP generated by a positive or negative corona discharge, or cometary discharge. After 15 min of exposure to NTP a significant decrease in the number of surviving spores in water suspensions was observed in all species. Complete spore inactivation and thus decontamination was observed in anthropophilic species after 25 min of exposure. Similarly, a significant decrease in the number of surviving spores was observed after 10-15 min of exposure to NTP on the surface of agar plates with full inhibition after 25 min in all tested species except of M. gypseum. Although the sensitivity of dermatophytes to the action of NTP appears to be lower than that of bacteria and yeast, our results suggest that NTP has the potential to be used as an alternative treatment strategy for dermatophytosis and could be useful for surface decontamination in clinical practice.

Nonthermal plasma--A tool for decontamination and disinfection.

By definition, the nonthermal plasma (NTP) is partially ionized gas where the energy is stored mostly in the free electrons and the overall temperature remains low. NTP is widely used for many years in various applications such as low-temperature plasma chemistry, removal of gaseous pollutants, in gas-discharge lamps or surface modification. However, during the last ten years, NTP usage expanded to new biological areas of application like plasma microorganisms' inactivation, ready-to-eat food preparation, biofilm degradation or in healthcare, where it seems to be important for the treatment of cancer cells and in the initiation of apoptosis, prion inactivation, prevention of nosocomial infections or in the therapy of infected wounds. These areas are presented and documented in this paper as a review of representative publications.

Treatment of a superficial mycosis by low-temperature plasma: a case report.

A case of dermatomycosis caused by zoophilic strain of Trichophyton interdigitale was treated by low-temperature plasma produced by direct current (DC) cometary discharge. The shortening of skin lesion persistence along with suppression of subjective discomfort and etiological agent was observed.

Immunomodulatory properties of subcellular fractions of a G+ bacterium, Bacillus firmus.

Mucosal immunization with non-living antigens usually requires the use of an adjuvant. The adjuvant activity of Bacillus firmus in the mucosal immunization of mice was described by our laboratory previously. In the present study, subcellular localization of B. firmus activities was followed. After mechanical disintegration, subcellular components of bacterium were fractionated by differential centrifugation and salting out. Bacterial cell walls, cytoplasmic membrane fraction, soluble cytoplasmic proteins, and ribosomal fractions were isolated. Their effect on the mouse immune system was studied. Lymphocyte proliferation and immunoglobulin formation in vitro were stimulated by bacterial cell wall (BCW), cytoplasmic membrane (CMF), and ribosomal fractions. BCW and CMF increased antibody formation after intratracheal immunization of mice with influenza A and B viruses, and increased protection against subsequent infection with influenza virus. The BCW fraction even induced intersubtypic cross-protection: Mice immunized with A/California/7/04 (H3N2) + BCW were resistant to the infection by the highly pathogenic A/PR/8/34 (H1N1) virus.