Bogumila Szponar

Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters

Background.  The lipopolysaccharide of Helicobacter pylori plays an important role in colonization and pathogenicity. The present study sought to compare structural and biological features of lipopolysaccharides from gastric and enterohepatic Helicobacter spp. not previously characterized.

Characterization of Lipopolysaccharides Present in Settled House Dust

ABSTRACT The 3-hydroxy fatty acids (3-OHFAs) in lipopolysaccharides (LPS) play an important role in determining endotoxin activity, and childhood exposure to endotoxin has recently been associated with reduced risk of atopic diseases. To characterize the 3-OHFAs in house dust (HD), we used gas chromatography-mass spectrometry to assay 190 HD samples. Dust from beds, bedroom floors, family rooms, and kitchen floors was collected as part of a birth cohort study of childhood asthma (study 1) and a longitudinal study of home allergen and endotoxin (study 2). We also measured endotoxin activity with a Limulus assay and computed specific activity (endotoxin activity per nanomole of LPS). Longer-chain (C16:0 and C18:0) 3-OHFAs were predominant in HD compared with short-chain (C10:0, C12:0, and C14:0) acids. Endotoxin activity was positively correlated with short-chain 3-OHFAs in both studies. In study 2, 3-OH C16:0 was negatively correlated and 3-OH C18:0 was not correlated with endotoxin activity, consistent with previous findings that the Limulus assay responds preferentially to LPS containing short-chain 3-OHFAs. Kitchen dust contained the highest concentrations of 3-OH C10:0, the highest endotoxin activities, and the highest specific activities (P < 0.03). Bed dust contained the largest amounts of long-chain 3-OHFAs, the highest concentrations of LPS, and the lowest specific activities. Apartments had significantly different types of LPS (P = 0.03) compared with single-family homes in study 2. These data suggest that the Limulus assay may underestimate exposure to certain types of LPS. Because nontoxic LPS may have immune modulating effects, analysis of 3-OHFAs may be useful in epidemiologic studies.

Distribution of 3-Hydroxy Fatty Acids in Tissues after Intraperitoneal Injection of Endotoxin

BACKGROUND 3-Hydroxy fatty acids (3-OH FAs) with 10- to 18-carbon chain lengths are constituents of the endotoxin [lipopolysaccharide (LPS)] of gram-negative bacteria. We investigated whether these FAs may be used as chemical markers in measuring endotoxin concentrations in mammalian tissue samples. METHODS We used gas-liquid chromatography-tandem mass spectrometry to measure 3-OH FAs in serum and tissues (heart, liver, and skeletal muscles) of rats after intraperitoneal injection of Escherichia coli LPS. One group of rats (group I) received a single LPS dose of 20 mg/kg of body weight; group II rats received the same total dose but over the course of 10 days (2 mg/kg each day). Rats receiving saline (group III) were used as controls. RESULTS 3-OH FAs with chain lengths of 10, 12, 14, 16, and 18 carbons were detected in all studied types of samples. Group I rats had 50-fold and group II rats had 3-fold higher serum concentrations of 3-hydoxytetradecanoic acid (3-OH 14:0, the predominant 3-OH FA of E. coli LPS) than group III rats. Concentrations of 3-OH 14:0 in livers from group I and II rats were similar and fourfold higher than in group III rats, whereas concentrations of the same acid in skeletal and heart tissues did not differ among the three groups of rats. 3-OH 14:0 dominated in heart and liver of group III rats, whereas 3-OH 16:0 (followed by 3-OH 14:0) dominated in skeletal muscles and blood. CONCLUSIONS 3-OH FAs 10-18 carbons in length, probably originating from endotoxin and mitochondrial beta-oxidation, are abundant in rat liver, skeletal muscles, and heart and can also be detected in blood. The widespread presence of these compounds in mammals limits their usefulness as LPS markers for endotoxin in clinical samples.

Protein Fraction of Barley Spent Grain as a New Simple Medium for Growth and Sporulation of Soil Actinobacteria.

A cheap value-added product, the protein fraction of barley spent grains is proposed as a source of a potential and economical cultivation medium. We showed that medium composed of protein fraction extract allows the isolation of actinobacteria, especially Streptomyces, from soil samples, and enhances the sporulation. It was used for the screening and production of the biologically active substances from actinobacteria.

Limitations in the use of 3-hydroxy fatty acid analysis to determine endotoxin in mammalian samples

3-Hydroxy fatty acids (3-OH FAs) of 10-18-carbon chain lengths are constituents of the lipopolysaccharide of Gram-negative bacteria. These acids are used as chemical markers for determining endotoxin in environmental samples. The present communication addresses the question whether this type of analysis also would be applicable to mammalian samples. Low levels (6.1+/-1.6-94.0+/-23.2 pmol/ml) of the studied 3-OH FAs were detected in blood from both conventional and germ-free rats. The levels were considerably higher (0.0-1.06+/-0.17 nmol/mg) in livers. The amounts of the 3-OH FAs did not differ between the two groups of rats. All analyses were made by gas chromatography-tandem mass spectrometry (GC-MSMS) for unequivocal identification. The results illustrate a limitation in using 3-OH FA analysis to determine endotoxin in mammalian samples since these acids may represent not only endotoxin but also products from mammalian mitochondrial fatty acid beta-oxidation.

Influence of Cu–Ti thin film surface properties on antimicrobial activity and viability of living cells

The paper describes properties of thin-film coatings based on copper and titanium. Thin films were prepared by co-sputtering of Cu and Ti targets in argon plasma. Deposited coatings consist of 90at.% of Cu and 10at.% of Ti. Characterization of the film was made on the basis of investigations of microstructure and physicochemical properties of the surface. Methods such as scanning electron microscopy, x-ray microanalysis, x-ray diffraction, x-ray photoelectron spectroscopy, atomic force microscopy, optical profilometry and wettability measurements were used to assess the properties of deposited thin films. An impact of Cu-Ti coating on the growth of selected bacteria and viability of the living cells (line L929, NCTC clone 929) was described in relation to the structure, surface state and wettability of the film. It was found that as-deposited films were amorphous. However, in such surroundings the nanocrystalline grains of 10-15nm and 25-35nm size were present. High surface active area with a roughness of 8.9nm, had an effect on receiving relatively high water contact angle value (74.1°). Such wettability may promote cell adhesion and result in an increase of the probability of copper ion transfer from the film surface into the cell. Thin films revealed bactericidal and fungicidal effects even in short term-contact. High activity of prepared films was directly related to high amount (ca. 51 %) of copper ions at 1+ state as x-ray photoelectron spectroscopy results have shown.

Heterogeneity in microbial exposure in schools in Sweden, Poland and Jordan revealed by analysis of chemical markers

We used gas chromatography–tandem mass spectrometry to analyze microbial components in 85 samples of airborne dust from schools in Jordan, Sweden, and Poland. To collect the samples, we allowed dust to settle on plexiglass plates hanging in the breathing zone in school buildings during both summer and winter. In each of the three countries, we conducted such sampling in two schools: one in an urban environment and the other in rural surroundings. The microbial marker profiles differed significantly between the schools and seasons. For example, samples from Jordan contained remarkably low levels of ergosterol (marker of fungal biomass) and high levels of 3-hydroxy acids (markers of lipopolysaccharide) of 10, 12, and 14 carbon chain lengths relative to such acids of 16 and 18 carbons in comparison with samples from Sweden and Poland. This dissimilarity in 3-hydroxy fatty acid distribution indicates significant differences in the populations of Gram-negative bacteria. We also noted that muramic acid (marker of bacterial biomass) exhibited the smallest variation between schools and seasons. In summary, our results demonstrate that exposure to microorganisms in indoor air in school buildings may differ markedly between countries, between seasons, and between urban and rural environments.

Bacterial and fungal markers in tobacco smoke

Previous research has demonstrated that cigarette smoke contains bacterial and fungal components including lipopolysaccharide (LPS) and ergosterol. In the present study we used gas chromatography-mass spectrometry to analyze tobacco as well as mainstream and second hand smoke for 3-hydroxy fatty acids (3-OH FAs) of 10 to 18 carbon chain lengths, used as LPS markers, and ergosterol, used as a marker of fungal biomass. The air concentrations of LPS were 0.0017 n mol/m(3) (N=5) and 0.0007/m(3) (N=6) in the smoking vs. non-smoking rooms (p=0.0559) of the studied private houses, and 0.0231 n mol/m(3) (N=5) vs. 0.0006 n mol/m(3) (N=5) (p=0.0173), respectively, at the worksite. The air concentrations of ergosterol were also significantly higher in rooms with ongoing smoking than in rooms without smoking. A positive correlation was found between LPS and ergosterol in rooms with smoking but not in rooms without smoking. 3-OH C14:0 was the main 3-OH FA, followed by 3-OH C12:0, both in mainstream and second hand smoke and in phenol:water smoke extracts prepared in order to purify the LPS. The Limulus activity of the phenolic phase of tobacco was 3900 endotoxin units (EU)/cigarette; the corresponding amount of the smoke, collected on filters from 8 puffs, was 4 EU/cigarette. Tobacco smoking has been associated with a range of inflammatory airway conditions including COPD, asthma, bronchitis, alveolar hypersensitivity etc. Significant levels of LPS and ergosterol were identified in tobacco smoke and these observations support the hypothesis that microbial components of tobacco smoke contribute to inflammation and airway disease.

An airborne actinobacteria Nocardiopsis alba isolated from bioaerosol of a mushroom compost facility

Actinobacteria are widely distributed in many environments and represent the most important trigger to the occupant respiratory health. Health complaints, including hypersensitivity pneumonitis of the workers, were recorded in a mushroom compost facility (MCF). The studies on the airborne bacteria were carried out to find a possible microbiological source of these symptoms. Culture analysis of compost bioaerosols collected in different location of the MCF was performed. An assessment of the indoor microbial exposure revealed bacterial flora of bioaerosol in the mushroom compost facility represented by Bacillus, Geobacillus, Micrococcus, Pseudomonas, Staphylococcus spp., and actinobacterial strain with white aerial mycelium. The thermotolerant actinobacterial strain of the same morphology was repeatedly isolated from many locations in MCF: air, compost sample, and solid surface in production hall. On the base of complex morphological, chemotaxonomic, and phylogenetic characteristics, the isolate has been classified as Nocardiopsis alba. Dominant position of N. alba in microbial environment of the mushroom compost facility may represent an indicator microorganism in compost bioaerosol. The bioavailability of N. alba in mushroom compost facility creates potential risk for the health of workers, and the protection of respiratory tract and/or skin is strongly recommended.

Assessment of microbial contamination within working environments of different types of composting plants

The objective of the study was to determine the degree of microbiological contamination, type of microflora, bioaerosol particle size distribution, and concentration of endotoxins in dust in different types of composting plants. In addition, this study provides a list of indicator microorganisms that pose a biological threat in composting facilities, based on their prevalence within the workplace, source of isolation, and health hazards. We undertook microbiological analysis of the air, work surfaces, and compost, and assessed the particle size distribution of bioaerosols using a six-stage Andersen sampler. Endotoxins were determined using gas chromatography–mass spectrometry (GC-MS). Microbial identification was undertaken both microscopically and using biochemical tests. The predominant bacterial and fungal species were identified using 16S rRNA and ITS1/2 analysis, respectively. The number of mesophilic microorganisms in composting plants amounted to 6.9 × 102–2.5 × 104 CFU/m3 in the air, 2.9 × 102–3.3 × 103 CFU/100 cm2 on surfaces, and 2.2 × 105–2.4 × 107 CFU/g in compost. Qualitative analysis revealed 75 microbial strains in composting plants, with filamentous fungi being the largest group of microorganisms, accounting for as many as 38 isolates. The total amount of endotoxins was 0.0062–0.0140 nmol/mg of dust. The dust fraction with aerodynamic particle diameter of 0.65–1.1 μm accounted for 28–39% of bacterial aerosols and 4–13% of fungal aerosols. We propose the following strains as indicators of harmful biological agent contamination: Bacillus cereus, Aspergillus fumigatus, Cladosporium cladosporioides, C. herbarum, Mucor hiemalis, and Rhizopus oryzae for both types of composting plants, and Bacillus pumilus, Mucor fragilis, Penicillium svalbardense, and P. crustosum for green waste composting plants. The biological hazards posed within these plants are due to the presence of potentially pathogenic microorganisms and the inhalation of respirable bioaerosol. Depending on the type of microorganism, these hazards may be aggravated or reduced after cleaning procedures. Implications: This study assessed the microbial contamination in two categories of composting plants: (1) facilities producing substrates for industrial cultivation of button mushrooms, and (2) facilities for processing biodegradable waste. Both workplaces showed potentially pathogenic microorganisms, respirable bioaerosol, and endotoxin. These results are useful to determine the procedures to control harmful biological agents, and to disinfect workplaces in composting plants.