The potential for use of non-thermal plasma in microbiology and medicine.

The overview provides basic information on non-thermal plasma, its properties, and methods of its generation. It gives examples of its use in the inactivation of bacteria including biofilms, fungi, and prions. Related applications in human medicine, namely in wound healing, antitumor therapy, dental medicine, and dermatomycosis therapy are also mentioned.

Comparison of fungicidal properties of non-thermal plasma produced by corona discharge and dielectric barrier discharge.

The inactivation of four micromycete species by action of non-thermal plasma was followed. Two sources of plasma were compared, namely, positive corona discharge and dielectric barrier discharge. The corona discharge appeared as suitable for fungal spore inactivation in water suspension, whereas the barrier discharge inactivated spores on the surface of cultivation agar. Cladosporium sphaerospermum was the most sensitive, being inactivated within 10 min of exposure to plasma, whereas Aspergillus oryzae displayed decrease in viable cell count only, the complete inactivation was not achieved even after 40 min of exposure. Intermediate sensitivity was found for Alternaria sp. and Byssochlamys nivea. The significant delay of growth was observed for all fungi after exposure to sublethal dose of plasma, but we failed to express this effect quantitatively.

The survival of micromycetes and yeasts under the low-temperature plasma generated in electrical discharge.

The fungicidal effect of low-temperature plasma generated by positive direct current discharge and its influence on the growth dynamics was evaluated on three micromycete species and yeast in water suspensions. The fungicidal effect was lower than analogous bactericidal effect and differs substantially among various fungal species. Together with the cidal effects, the slower growth of exposed fungal spores was observed.

Stimulation of protective and cross-protective immunity against influenza B virus after adjuvant mucosal immunization of mice.

Intratracheal immunization of mice with inactivated influenza B virus and delipidated Bacillus firmus as adjuvant increases protection of mice against infection with the homologous virus strain and induces cross-protection: mice immunized by influenza virus B/Yamanashi 166/98 were protected even against phylogenetically distant virus drift variant B/Lee/40 lethal for mice.

Decontamination effects of low-temperature plasma generated by corona discharge. Part I: an overview.

The first part of this article is devoted to a minireview of basic terms of plasma physics and chemistry described in a way understandable even to nonspecialists in the field. Among the methods of generation of low-temperature plasma, the unipolar electric corona discharge in air at atmospheric pressure is described in more detail. Selected studies are mentioned that concern the decontamination effects of low-temperature plasma and the currently known mechanisms of its microbicidal action. The key part of this mechanism is the action of UV light and both charged and uncharged particles. The most important common mechanism seems to be different forms of reactive oxygen species and some methods of their determination are therefore briefly described.

Decontamination effects of low-temperature plasma generated by corona discharge. Part II: new insights.

The second part of our paper presents the results of experiments with the decontamination of surfaces by low-temperature plasma generated by corona discharge in air at atmospheric pressure. A simple device is described and the effects of the corona discharge on model microorganisms, viz. the yeast Candida albicans, Gram-negative bacteria Escherichia coli, Enterobacter aerogenes, Neisseria sicca, Stenotrophomonas maltophilia, Gram-positive bacteria Deinococcus radiodurans, Enterococcus faecium, Staphylococcus epidermidis, Streptococcus sanguinis, and vegetative and spore forms of Geobacillus stearothermophilus are discussed. A similar microbicidal effect after about one-minute exposure was observed in all vegetative forms of the microorganisms. Measurement in growth inhibition zones on a semisolid medium was used to determine the dependence of the microbicidal effect on exposure time and the distance between electrodes. Counting of colonies served to assess the microbicidal effect of the discharge on contaminated inert surfaces observable after more than 1 min exposure. Geobacillus stearothermophilus spores were found to have several times lower susceptibility to the action of the discharge and the microbicidal effect was observed only after an 8 min exposure. Reaction with the iodide reagent did not unambiguously demonstrate the difference between ozone and singlet oxygen as presumed active components of the corona. The area distribution of reactive oxygen species was determined; it was found to differ from the Wartburg law depending on exposure time. Qualitative evidence was obtained on the penetration of the reactive oxygen species into the semisolid medium.

Chromatographic analysis in bacteriologic diagnostics of blood cultures, exudates, and bronchoalveolar lavages.

This article summarizes our previously achieved and published results. The method for the determination of bacterial volatile fatty acid patterns (VFA) in clinical samples was elaborated. It employs gas chromatography (GC), solvent extraction or head-space solid phase microextraction (SPME). This method was validated by analyses of reference bacterial strains. After cultivation in defined media, aerobic and facultative anaerobic bacteria provided profiles with a low or none acid content, while anaerobic bacteria provided characteristic but medium-dependent profiles with a higher acid content. This method was used for the analyses of clinical samples of total 375 blood cultures, 205 suppurative and apyogenous exudates, and 210 bronchoalveolar lavages (BALs). These analyses enabled within 30 minutes the detection of microbes, probably non-sporulating anaerobes not found by false-negative cultivation, in 11.2% of blood cultures, in 20.0% of exudates, and in 9.0 to 20.0% of BALs. Using the mass spectrometry (MS) methods, a number of other components with unclear diagnostic importance were found in BAL samples, in particular hydrogen cyanide, methanol, ethanol, hexanol, acetone, cyclohexanone, acetonitrile, formaldehyde, acetaldehyde, ethyl acetate, and other esters. Cyclohexanone, occurring mainly in BALs of patients with pneumonia, undergoing intensive care, may originate as a residual solvent from the plastic parts of the ventilation apparatus.

Intratracheal and intranasal immunization with ovalbumin conjugated with Bacillus firmus as a carrier in mice.

Inactivated Bacillus firmus (BF), G+ nonpathogenic bacterium of the external environment, was coupled to ovalbumin (OVA) and used in immunization experiments as antigen carrier. Balb/c mice were immunized thrice intra-tracheally and intra-nasally with conjugates of OVA and BF. Surprisingly, administration of OVA-BF conjugates inhibited anti-OVA IgG response in both sera and mucosal secretions if compared to an exposure to OVA alone. The suppression of antigen-specific antibody production was accompanied by promotion of TH1 phenotype.

Immune response after adjuvant mucosal immunization of mice with inactivated influenza virus.

Satisfactory mucosal immunity in the respiratory tract is very important for protection against influenza. It can be achieved only by mucosal immunization. Mucosal vaccination with inactivated influenza virus may not be sufficiently effective and suitable adjuvants are therefore sought. We tested intratracheal immunization of mice with inactivate B type influenza virus in a mixture with formolized G+ bacterium Bacillus firmus, whose adjuvant effects have previously been documented in another system. The treatment resulted in a marked increase of both systemic and mucosal antibody response in IgG and IgA classes. Stimulation of T lymphocytes after adjuvant immunization was very mild, no proliferation taking place after specific stimulation with antigen in vitro. However, slightly increased systemic (spleen) and local (lungs) production of cytokines without perceptible Th1/Th2 polarization was determined. B. firmus is an efficient adjuvant in respiratory tract immunization while with subcutaneous immunization it lowers the antibody response.

Immunostimulatory effect of Bacillus firmus on mouse lymphocytes.

Bacillus firmus (a Gram-positive nonpathogenic and harmless bacterium), was shown to be a strong polyclonal activator of mouse B lymphocytes as estimated by ELISA testing of Ig concentrations in culture supernatants after incubation of BALB/c mouse splenocytes with inactivated bacillus. Synthesis of all main Ig classes and all IgG subclasses was stimulated in vitro, the considerable effect on IgA formation being the most interesting feature. B cell stimulation was T cell dependent, as was demonstrated by the effect of B. firmus on all Ig isotypes and by comparison of lymphocyte response of nu/nu mice and heterozygous nu/+ mice. The effect of B. firmus on splenocyte proliferation was stimulatory or suppressive depending on the dose of the bacterium. Increased synthesis of IFN-gamma and IL-10 (detected by ELISA in splenocyte culture supernatants) showed probable stimulation of Th1 and Th2 subpopulations. Considering the stimulatory effect on IgA formation and macrophage stimulation, B. firmus seems to be a prospective mucosal adjuvant and/or probiotic.

Blood cultures evaluation by gas chromatography of volatile fatty acids.

The volatile fatty acid profiles were determined by a simple gas chromatographic method in 375 microbiologically positive and negative blood cultures. Aerobic bacteria yielded profiles with low content of acids, some of which were chromatographically negative. Anaerobic bacteria produced more acids and more distinctive profiles. The method makes it possible to confirm the microbiological findings and to select the blood cultures containing anaerobic bacteria within 30 minutes.

Polyclonal activation of human lymphocytes by Bacillus firmus and its constituents.

Bacillus firmus strongly stimulates Ig synthesis in the cultures of human peripheral blood mononuclear leukocytes. As apparent from the character of Ig formation and blastic transformation, the stimulation has features of a polyclonal activation of B lymphocytes without substantial participation of T lymphocytes. B firmus is a strong B cell polyclonal activator even for human cord blood lymphocytes. The most striking feature is the strong stimulation of IgA synthesis in both adult and cord blood lymphocytes. Several crude fractions were isolated from B. firmus. None of them exhibited any remarkable enhancement of activity but the cytoplasmic fraction P-40 was clearly more potent than the intact bacilli. On the other hand, cell wall peptidoglycan, a well known polyclonal activator of B cells, had a much lower activity than whole bacteria. The effect of B. firmus on the stimulation of Ig formation is thus relatively complex; it is not caused mainly by peptidoglycan but rather by some cytoplasmic constituents of the bacterium.

The influence of Propionibacterium acnes (Corynebacterium parvum) fractions on immune response in vivo.

Bacterin of Propionibacterium acnes (Corynebacterium parvum), its cellular fractions (lipids, fractions obtained by mechanical disruption and differential centrifugation, by phenol-water and pyridine extractions), and a polysaccharide from culture filtrate were prepared and tested in mice. The activation of RES by splenomegaly and hepatomegaly, prevention of listerial infection, prevention of the lethal effect of sarcoma 180, and depression of liver microsomal cytochrome P-450 were employed. The bacterin was effective in all tests. Lipid-free cells were less active, in particular in the activation of RES and in the listerial infection model. Fractions prepared by the disruption and differential centrifugation lost their activity in all tests along with a decrease in molecular weight. Lipids extracted by ethanol caused pronounced splenomegaly and decreased the cytochrome P-450 content. The residue left after the phenol-water extraction was very active, its delipidation did not destroy the activity. Pyridine extraction provided a completely inactive extract, but a very active residue. The possibility of reducing the complexity of bacterin while preserving immunomodulatory effect is demonstrated.

Resistance to infection and activation of the monocyto-macrophage system caused by Bacillus firmus and its fractions.

Crude lipids isolated from Bacillus firmus, but not from other bacilli, were previously found to induce significant resistance against Listeria monocytogenes infection in mice. In this study, formaldehyde- and heat-killed bacterins of eight Bacillus species and some cellular fractions of B. firmus were prepared and tested for further immunomodulatory activities. Crude lipids, their aqueous extract, LTA, Protodyne and Pex-residue preparations exhibited a strong anti-infection activity, whereas Pextract, P40 and all bacterins tested had no effect. Formaldehyde-killed bacterins, live bacteria and the P40 preparation of both B. firmus strains, as well as bacterins of both B. subtilis strains, induced pronounced splenomegaly in mice. Peptidoglycan and Pex-residue induced significant depression of cytochrome P-450 in mouse liver microsomes after application of 0.1 mg per mouse. Optimal conditions for obtaining a bacterial suspension exhibiting these immunomodulatory properties were elaborated.

Effect of Bacillus firmus and other sporulating aerobic microorganisms on in vitro stimulation of human lymphocytes. A comparative study.

B. firmus activates human peripheral blood lymphocytes in vitro. Bacteria inactivated by heat or by formaldehyde were about equally effective, stimulating the blastic transformation of lymphocytes at doses of 10-200 mg/L and Ig formation in the culture at 10-500 mg/L. The action of formaldehyde treated B. firmus was compared with that of analogously inactivated B. subtilis, B. polymyxa, B. coagulans, B. megaterium, B. pumilus, B. cereus and B. lentus at a concentration of 100 mg/L. All these bacilli mildly stimulated blastic transformation and most of them substantially stimulated Ig formation, but B. firmus was the most efficient in stimulating the formation of Ig of all classes, in particular IgM and IgA. Its effect on Ig formation was comparable with that of PWM and was unusually high as compared with that of other bacteria. B. firmus is apparently a strong polyclonal activator of B lymphocytes. Its cells or their components could be potentially used for modulating immune reactions.

Composition of lipid A in the S and R forms of Shigella dysenteriae serovar 1.

A chemical analysis was performed of lipid A, isolated by acid hydrolysis of the lipopolysaccharide from the S and R forms of Shigella dysenteriae serovar 1. Differences in the moiety of both lipids and sugars were compared. The lipid portion consisted of a homologous series of fatty acids ranging from C12:0 to C18:0 (predominant homologues, C12:0, C14:0 and C16:0) and the homologous series of 3-hydroxy acids ranging from C12:0 to C16:0 (predominant homologue, 3-OH-C14:0). The major sugar portion consisted of D-glucosamine. The toxicity of lipid A in mice (LD50) ranged between 300-400 micrograms/mouse, and values from the Limulus amebocyte lysate assay were recorded as titres of 10(-5) to 10(-6) mg/ml.

Effect of crude bacterial lipids on the course of Listeria infection in mice.

Crude lipids from 37 strains belonging to 32 bacterial species were isolated. By injecting mice with lipids 5 d prior to challenge with a virulent strain of Listeria monocytogenes, immunostimulatory activity in 19 preparations was found. In general, lipids of Gram-negative bacteria appeared to be more effective. As to bacilli, an extraordinary activity was found in the lipids of Bacillus firmus. Lipids of various species of the genus Listeria were found to be active in approximately one-half of cases. Among other Gram-positive bacteria, significant activity of lipids was found in Corynebacterium xerosis, Propionibacterium acnes and BCG. The composition of fatty acids in the lipids did not differ significantly from that reported in the literature and their mutual differences could not explain the different biological activity. In selected strains of Gram-negative bacteria lipids were repeatedly purified with anhydrous chloroform; these preparations were found to be inactive as compared with original chloroform-methanol lipids.

Cellular fatty acids and fatty aldehydes of Listeria and Erysipelothrix.

The cellular fatty acid composition determined by gas chromatography-mass spectrometry, was found not to differ among Listeria monocytogenes, L. innocua and L. ivanovii. Slight quantitative differences found in the fatty acid pattern of L. welshimeri were significantly pronounced in L. denitrificans. L. murrayi and L. grayi displayed characteristic closely related patterns. Considerable amounts of fatty aldehydes and their dimethyl acetals were observed in hydrolysates and methanolysates of L. seeligeri. The fatty acid composition of Erysipelothrix rhusiopathiae was found to strongly differ from that of Listeria.

Membrane fluidity in Bacillus subtilis. Validity of homeoviscous adaptation.

The validity of the principle of homeoviscous adaptation for Bacillus subtilis was tested by comparing fluorescence anisotropy (1,6-diphenyl-1,3,5-hexatriene) and electron-spin resonance (16-doxylstearate) measurements carried out in isolated plasma membranes and in phospholipid fractions. The physical measurements were supplemented by fatty-acid analysis. The results support our previous findings on intact cells. The thermoadaptive mechanism of B. subtilis manifested as an increase in relative proportion of branched anteiso-C15 and anteiso-C17 fatty acids, are not strong enough to compensate for the marked physical change of membrane fluidity induced by temperature decrease.

Fatty acid composition of a Propionibacterium acnes vaccine and its relationship to immunostimulatory activity.

The distribution of lipids of Propionibacterium acnes (Corynebacterium parvum) vaccine strain in chloroform-methanol, ethanol and light petroleum extracts was determined. Firmly bound lipids released by hydrolysis were also investigated. The petroleum extract differs from other lipidic fractions in its fatty acid composition. The presence of linolic, tuberculostearic and 10-ketostearic acids and branched fatty alcohols was observed in addition to previously described fatty acids of P. acnes. Changes in fatty acid composition during growth of the vaccine strain were determined and extremes were found at 95 h of cultivation. These extremes coincided with a maximum in the immunostimulatory efficiency as measured by the spleen enlargement test. The biological activity of static and stirred cultures of the vaccine strain was in correlation with their fatty acid composition.