J. Julák

Evaluation of exudates by solid phase microextraction–gas chromatography

Head-space solid phase microextraction combined with gas chromatography (SPME-GC) was used for the determination of bacterial volatile fatty acid (VFA) patterns. The method was validated with cultures of reference bacterial strains. It was confirmed that VFA production depends on the composition of the cultivation medium, which limits accurate characterisation of particular bacterial species. A set of 195 clinical exudates of various origin and consistence was analysed using SPME-GC and compared with 73 samples extracted using tert-butyl methyl ether. Approximate agreement of VFA profiles with cultivation findings was found in most cases. However, 20.5% of clinical exudates with distinct VFA profiles appeared to be false-negative by cultivation. Using SPME-GC of exudates, the frequency of false-negative cultivations was higher than that of solvent extraction of exudates or blood cultures found previously. The described method is suitable for preliminary detection of bacteria, particularly non-sporulating anaerobes, in clinical samples. It can reveal false-negative findings due to cultivation. Analysis can be performed in 30 min without the need for cultivation.

Effect ofBacillus firmus and other sporulating aerobic microorganisms onin vitro stimulation of human lymphocytes. A comparative study

B. firmus activates human peripheral blood lymphocytesin 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 treatedB. firmus was compared with that of analogously inactivatedB. subtilis, B. polymyxa, B. coagulans, B. megaterium, B. pumilus, B. cereus andB. lentus at a concentration of 100 mg/L. All these bacilli midly stimulated blastic transformation and most of them substantially stimulated Ig formation, butB. 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.

Membrane fluidity inBacillus subtilis. Validity of homeoviscous adaptation

The validity of the principle of homeoviscous adaptation forBacillus subtilis was tested by comparing fluorescence aniaotropy (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 ofB. subtilis manifested as an increase in relative proportion of branchedanteiso-C15 andanteiso-C17 fatty acids, are not strong enough to compensate for the marked physical change of membrane fluidity induced by temperature decrease.

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 stimulatedin 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 ofB. firmus on all Ig isotypes and by comparison of lymphocyte response of nu/nu mice and heterozygous nu/+mice. The effect ofB. firmus on splenocyte proliferation was stimulatory or suppressive depending on the dose of the bacterium. Increased synthesis of IFN-γ 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.

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

Crude lipids isolated fromBacillus firmus, but not from other bacilli, were previously found to induce significant resistance againstListeria monocytogenes infection in mice. In this study, formaldehyde-and heat-killed bacterins of eightBacillus species and some cellular fractions ofB. 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 bothB. firmus strains, as well as bacterins of bothB. 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.

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.

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.

Plasma Jetlike Point-to-Point Electrical Discharge in Air and Its Bactericidal Properties

A new type of point-to-point plasma jetlike electric discharge at atmospheric pressure in air without outsource gas flow was observed. Some of its properties were studied, including its bactericidal properties.

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.

Adjuvant effect of Bacillus firmus on the expression of cytokines and toll-like receptors in mouse nasopharynx-associated lymphoid tissue (NALT) after intranasal immunization with inactivated influenza virus type A

Due to the persisting threat of development of new highly pathogenic influenza A subtypes, a mucosal vaccination which would induce a potent and cross-protective reaction is desirable. We succeeded in mucosal immunization of mice with an inactivated influenza A virus by using delipidated Bacillus firmus (DBF) as adjuvant. The mechanism of adjuvant effect was followed in NALT by comparing the response after intranasal immunization by inactivated influenza virus type A (H1N1) alone, adjuvant alone (DBF), or by a mixture of virus+DBF. Expression of selected gene groups was tested via qPCR at 7 different time-points: cytokines (IL-2, IFN-γ, IL-4, IL-6, and IL-10), type I interferons (IFN-α4, IFN-α11, IFN-α12, and IFN-β), toll-like receptors (TLR2, TLR3, TLR7, and TLR9), iNOS and CCR7. Intranasally administered DBF and the mixture of virus+DBF induced an elevated expression of IFN-γ, IL-6 and IL-10 cytokines, type I interferons, iNOS, and pDC markers in NALT. Multimarker qPCR data was analyzed by relative quantification and by principal component analysis. DBF has been shown to be a very efficient adjuvant for the stimulation of innate immunity after IN immunization. DBF accelerated, increased, and prolonged the antiviral response.