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PubMed2002

[In vitro selection and affinity function of the aptamers to Bacillus anthracis spores by SELEX].

To obtain oligonucleotide aptamers, specifically binding to Bacillus anthracis spores, and to find the relationship between the structures and the affinities, and to determine whether the aptamers can be used as a novel molecule for spore detection, a synthetic 35 mer random DNA library was subjected to 18 rounds of selection by using SELEX (systematic evolution of ligands by exponential enrichment) protocol against spores of Bacillus anthracis vaccine strain A. 16R. The selected aptamers were cloned and sequenced. Software packages CLUSTALX (1.8) and DNASIS v2.5 were employed to analyze the conserved sequences and second structures of the aptamers, respectively. Affinities of aptamers to the spores were visualized by biotin streptavidin horseradish peroxidase system. DAB was used to visualize signals, as an assay method. A membrane-based hybrid sandwich assay was developed for detecting Bacillus anthracic spores by using a 5'-biotinylated ssDNA aptamers and anti-spore antibodies. PCR amplification band pattern of the first round selection was different from that of the ninth round. The binding assay demonstrated that the affinity of the eighteenth round pool increased thirty-seven folds more than that of the first round pool. The affinities of the aptamers were different: the highest A at 450 nm was 1.20, and the lowest was 0.20. The secondary structure analysis revealed possible stem-loop and hairpin structures for binding to the spores. The colorimetry on the immuno-membrane got the best signal with a ratio of 16 microgram aptamer to 4x10(7) spores. A set of aptamers with considerable binding affinity to Bacillus anthracis spores was successfully selected from the initial random ssDNA pool. The stem-loop and hairpin at 5' end of the aptamers worked as the main motif in the interaction between oligonucleotides and spores, while the neighbor bases of the triple structure might affect the stability. Therefore ssDNA aptamers seem to be a type of potential diagnostic molecule.

Bacillus anthracis↗
PubMed1992

Alternaria spore and mycelium sensitivity in allergic patients: in vivo and in vitro studies.

Extracts from Alternaria spores and mycelia were prepared to evaluate their allergenic potencies in allergic patients. Twelve Alternaria-sensitive patients, with histories of rhinitis or asthma, were submitted to skin prick tests and five of 12 patients received nasal challenges with spores and mycelia. In vitro, the allergenic activity of each extract was determined by RAST, basophil histamine-release and RAST-inhibition. All patients demonstrated skin reactivity to both extracts while skin reactivity to mycelia was higher than that induced by the spore extract (P < .005). Of the 12 patients, 11 had positive mycelia-RAST and 9/12 had positive spore-RAST. It was found that mycelium-IgE antibody levels were higher than spore-IgE antibody levels (P < .005). Nine RAST-positive patients had positive histamine release tests (> 50%) and basophils challenged with mycelia appeared 10-fold more sensitive compared to the spore challenge. In four of five patients subjected to nasal provocation tests, immediate-type rhinitis was elicited either after spore or mycelium challenge. The patients exhibited a higher nasal reactivity with the mycelium challenge than with spores. RAST-inhibition studies demonstrated that mycelial extracts shared common allergens with spore. These results indicated that Alternaria spore and mycelium were potent allergens in allergic patients and there was a variability in the pattern of in vivo and in vitro reactions between the patients for each allergen.

Adolescent↗
PubMed2004

Atmospheric concentrations of Cladosporium spp. and Alternaria spp. spores in Zagreb (Croatia) and effects of some meteorological factors.

The aim of the study was to analyse the relationship between meteorological conditions and Alternaria and Cladosporium spore concentrations in the air of Zagreb in August 2002 and August 2003. These months were chosen because they represented climatic extremes. A 7-day VPPS 2000 Hirst volumetric pollen and spore trap was used for spore sampling. Spores marked as 'others' (ascospores, basidiospores, Epicoccum, Ustilago, Pithomyces, Helminthosporium, Stemphylium, Torula, Botrytis, Didymella) were found to have predominated in the month of August in both 2002 and 2003 with 91.1% and 70.5%, respectively. Because of favourable weather conditions (higher air temperature and minimal precipitation) in August 2003, the concentrations of Alternaria and Cladosporium spores were 3.4-fold those recorded in the same month in 2002. Also, the peak daily concentrations of these spores were measured on days without precipitation and with higher air temperature. Intradiuranal variation in the Alternaria and Cladosporium spore concentrations was identical in 2002 and 2003 (lowest in 2-hour interval between 06:00-08:00, and highest between 10:00-12:00). In spite of the three-fold increase in the Cladosporium spore concentration in August 2003, the borderline concentration of 3,000 spores/m3 air that is associated with the occurrence of allergic reactions was only exceeded on a single day. Air concentration of Alternaria spores exceeded borderline value of 100 spores/m3 air on as many as 17 days, suggesting that at that time of the year the risk of allergic reaction was only present in individuals allergic to this spore type.

Alternaria↗
PubMed1989

[Comparison of the sporocidal effects of two different disinfectants i a model experiment with pathogenic and nonpathogenic clostridial spore as well as with Bacillus cereus].

Official methods concerning the examination of sporocidal effects of disinfectants are presently lacking in the Federal Republic of Germany. Spore resistance of selected clostridial species towards formaldehyde and peracetic acid was examined by suspension tests and germ-carrier tests in comparison to spores of Bacillus cereus. Formaldehyde in concentrations between 5% and 20% as well as peracetic acid in concentrations between .004% and .125% were tested. Spore concentration of 10(6) spores/ml in the suspension-tests and 10(4) spores/ml in the germ-carrier tests with wooden germ-carriers were used. Against formaldehyde spores of Cl. septicum had the lowest resistance in the suspension tests followed by Cl. difficile, Cl. tertium, Cl. bifermentans and Cl. perfringens. Cl. sporogenes strain NCTC 532 had the most resistant spores. Concerning peracetic acid the situation was nearly the same. The resistance of all spores of pathogenic species under investigation towards chemical disinfectants was covered by those of Cl. sporogenes NCTC 532 treated with isopropanol. Isopropanol treated spores of B. cereus offer an alternative solution for practical disinfectant testing because spore production as well as growth of clostridia entail great expense. In order to cover the resistance of spores of Cl. perfringens A, the most resistant pathogenic clostridial species tested here, increased spore concentrations of B. cereus would be necessary.

Bacillus cereus↗
PubMed1979

Exposure of rabbits to spores of Aspergillus fumigatus or Penicillium sp: survival of fungi and microscopic changes in the respiratory and gastrointestinal tracts.

Rabbits were exposed to spores of Aspergillus fumigatus by 1 of 2 routes: exposure to aerosols of dry spores or introduction of liquid suspensions of spores directly into the stomach. Rabbits also were exposed to aerosols containing spores of a Penicillium sp. Cultural and microscopic examinations of tissues from the respiratory and gastrointestinal tracts indicated fungi were distributed throughout the gastrointestinal tract of the rabbits within 1 hour after exposure to aerosols of A fumigatus or Penicillum spores. Viable A fumigatus and Penicillium were detected in lung tissues of rabbits for 2 or 3 weeks after inhalation of spores. Aspergillus fumigatus was isolated from the gastrointestinal tract no more than 1 week after aerosol exposure, and Penicillium, not beyond 48 hours. However, when large numbers of A fumigatus spores were introduced directly into the stomach, fungi were isolated from tissues for as long as 16 days after exposure even though the intestinal contents were negative 4 to 7 days after introduction of spores. Tests for precipitating antibody were negative, with one exception, among 26 rabbits surviving for 2 weeks or more. Microscopic changes were more pronounced in rabbits exposed to spores of A fumigatus than in rabbits exposed to Penicillium spores.

Animals↗
PubMed1981

Isolation of spore specific allergens from Alternaria.

Extracts of Alternaria alternata spores and mycelia were prepared. Crossed immunoelectrophoresis showed the presence of at least 40 antigens in these extracts. Analysis of seven commercial extracts by CIEP and isoelectric focusing showed many fewer components and a high degree of variability. Eight of the antigens in the spore extract demonstrable by CIEP were not present in mycelia extracts. This spore-specific fraction was isolated by solid phase immunoadsorption and shown to be potent antigenically and allergenically. The extracts of mycelia, spore and spore-specific fraction demonstrated strong reactivity with 92 Alternaria sensitive patients by both RAST and skin test. About half of the patients were significantly more reactive to spore and/or spore-specific than the mycelia. Many of the spore antigens are either common to or cross-reactive between spore and mycelia but there are a minimum of eight spore-specific antigens, at least one of which is a strong allergen.

Allergens↗
PubMed1987

Effects of sawdust pollution on the germination of fungal spores in Lagos Lagoon.

Four fungi, Aspergillus flavus, Aspergillus giganteus, Cladosporium oxysporum a and Trichoderma aureoviride, isolated from Lagos Lagoon, were tested for spore germination in aqueous sawdust extract of different hardwood species (Khaya ivorensis, Mitragyna ciliata and Triplochiton scleroxylon). Extracts of M. ciliata were inhibitory to spore germination especially at higher concentrations. Germ tubes of spores decreased in length with increase in extract concentration for C. oxysporum and T. aureoviride, while Aspergillus species showed increase in length up to a peak at 3% sawdust extract concentration, and thereafter germ tube lengths decreased with increasing concentration. Extracts of T. scleroxylon stimulated the spores and percentage germination increased at higher extract concentrations with no significant difference in germ tube length. Similarly, K. ivorensis had a stimulatory effect on spore germination and length of germ tube, especially at higher extract concentrations. Addition of soluble exogenous carbon and nitrogen sources to sawdust extract enhanced spore germination. Apart from A. flavus, which had only 9% germination, all the spores failed to germinate in lagoon water having 21% salinity. The spores were also inhibited by in lagoon water having 21 per thousand salinity. The spores were also inhibited by various inorganic salts, i.e. CaCl(2), KH(2)PO(4), MgSO(4) and NaCl, present in Lagos Lagoon. The results suggest that sawdust pollution causes enrichment of the lagoon, thereby enhancing spore germination.

Journal Article↗
PubMed1971

Coordinative binding of divalent cations with ligands related to bacterial spores. Equilibrium studies.

It has been repeatedly postulated that the high heat resistance of bacterial spores is due to stabilization of biopolymers in the spore interior by a solid deposit of protective cement consisting of coordination complexes of ligands with divalent metal ions. This report presents data on metal-binding characteristics of some of the ligands related to spores as determined by means of potentiometric equilibrium measurements under conditions of temperature and ionic strength (t = 25.0 degrees C; mu = 1.0 KNO(3)) identical with those reported earlier by the authors in order to facilitate correlation by using comparable data. The spore ligands investigated in this study included 2,6-pyridinedicarboxylic acid (DPA), alpha,epsilon-diaminopimelic acid, D-glutamic acid, and D-alanine in a ratio of 1:1 with metal ions which are known to play a role in heat resistance of spores. Stability constants of the chelates of these spore ligands with metal ions such as Ca(II), Mg(II), Cu(II), Ni(II), Zn(II), Co(II), and Mn(II) have been determined. In general the metal chelates of DPA exhibited the greatest stability. On the basis of a consideration of the stability data together with the known configurations of the ligand and the coordination requirements of the metal ions, possible structures indicating the coordinate binding of the spore ligands with the metal ions are presented. All the metal chelates except those of Ca(II) were found to undergo hydrolysis and separation of solid phase in the pH range 7-8.5. The relatively greater hydrolytic stability of Ca(II) chelates and the high affinity of DPA for metal ions appear to be of biological significance insofar as these two spore components are more widely associated with the heat resistance of bacterial spores.

Alanine↗
PubMed2004

The high-resolution architecture and structural dynamics of Bacillus spores.

The capability to image single microbial cell surfaces at nanometer scale under native conditions would profoundly impact mechanistic and structural studies of pathogenesis, immunobiology, environmental resistance, and biotransformation. Here, using in vitro atomic force microscopy, we have directly visualized high-resolution native structures of bacterial endospores, including the exosporium and spore coats of four Bacillus species in air and water environments. Our results demonstrate that the mechanisms of spore coat self-assembly are similar to those described for inorganic and macromolecular crystallization. The dimensions of individual Bacillus atrophaeus spores decrease reversibly by 12% in response to a change in the environment from fully hydrated to air-dried state, establishing that the dormant spore is a dynamic physical structure. The interspecies distributions of spore length and width were determined for four species of Bacillus spores in water and air environments. The dimensions of individual spores differ significantly depending upon species, growth regimes, and environmental conditions. These findings may be useful in the reconstruction of environmental and physiological conditions during spore formation and for modeling the inhalation and dispersal of spores. This study provides a direct insight into molecular architecture and structural variability of bacterial endospores as a function of spatial and developmental organizational scales.

Bacillus↗
PubMed1990

Spore coat is altered in modB glycosylation mutants of Dictyostelium discoideum.

The modB mutation eliminates specific carbohydrate epitopes from glycoproteins which are expressed primarily in prespore and spore cells of differentiating Dictyostelium discoideum. Spores formed by the mutant show several phenotypes. Whereas mutant spores germinate efficiently after heat activation, they germinate poorly after urea activation. Following germination, at least one glycosylation-defective glycoprotein is cleaved, and the larger fragment is released in soluble form from the spore coat. However, an earlier difference in the spore coat can be traced back to the nongerminated spore coat, as detected by the elutability of protein from intact spores by chemical extraction. An altered character of the pregermination spore coat is also suggested by increased labeling by a fluorescent lectin which binds to its interior. The findings are consistent with a change in the character of certain molecular contacts leading to altered characteristics of the mutant spore coat, which are specific because they are distinctive from changes observed in another glycosylation mutant which affects a different epitope.

Dictyostelium↗
PubMed1991

The effects of spore loading on the growth of Penicillium chrysogenum immobilised in kappa-carrageenan.

Penicillium chrysogenum spores were immobilised in kappa-carrageenan. The effect of the number of viable spores immobilised per bead on the rate of germination and degree of subsequent mycelial growth was investigated. The distribution of active mycelium throughout the bead was determined. At a high spore loading (10(3)-10(4) viable spores per bead) the biomass concentration was low and the majority of the actively respiring biomass was located at the bead periphery. Reducing the spore loading (to 50 viable spores per bead) resulted in a fourfold increase in immobilised biomass concentration. At very low spore loadings (5 and 10 viable spores per bead) the concentration of biomass decreased, but mass transfer throughout the bead improved and the uniformity of active immobilised biomass increased. The spore loading also affected the morphology of the growing hyphae and the extent of free cell growth.

Carrageenan↗
PubMed1994

Structural roles of the spore coat proteins in Dictyostelium discoideum.

The integrity of spores formed by mutant strains of Dictyostelium discoideum lacking the major spore coat proteins, SP96, SP70, or SP60, was compared to that of wild-type strains. Single, double, and triple knock-out strains developed normally and produced spores which were indistinguishable from wild-type spores by light or electron microscopy. However, the mutant strains were susceptable to staining with the lectin, ricin A, which recognizes a galactose-rich polysaccharide that is normally hidden by overlying spore coat proteins. The intensity of staining with fluorescently labeled ricinA increased as the spore coat proteins were incrementally lost. While these results indicate that the major outer spore coat proteins are not essential for the construction of a multi-layered spore coat in Dictyostelium, they show that the spores are more porous which might make them at risk to predators before germination.

Animals↗
PubMed1993

Expression of two cloned mRNA sequences during development and germination of spores of the sensitive fern, Onoclea sensibilis L.

A library of complementary DNA (cDNA) clones has been prepared from poly(A)+RNA of spores of the sensitive fern, Onoclea sensibilis L. By differential hybridization with labeled probes made to poly(A)+RNA of spores, gametophytes and leaves, two spore-specific clones (pOSS68 and pOSS194) were selected and characterized. Northern blot analysis showed that RNA sequences homologous to the two cDNA clones first appear in the post-meiotic spore and increase in abundance during spore maturity. Both RNA sequences decay during photoinduced germination of the spores and do not reappear in the gametophytes. In spores imbibed in the dark under conditions which do not favor germination, no significant decrease in pOSS194-mRNA abundance is noted. In contrast, the decrease in pOSS68 mRNA in dark-imbibed spores parallels that observed in photoinduced spores. The predicted amino-acid sequence of pOSS194 has a striking similarity to the early light-inducible proteins expressed during the greening of etiolated pea and barley seedlings, whereas that of pOSS68 shows some homology to proteins encoded by late-embryogenesis-abundant mRNAs of angiosperm embryos.

Base Sequence↗
PubMed1989

The preferential inhibition of Bacillus subtilis spore outgrowth by chloroquine.

Chloroquine inhibited the outgrowth of Bacillus subtilis spores at a 10-fold lower concentration than that required to prevent vegetative growth. Analysis of macromolecular synthesis in outgrowing spores and vegetative cells in the presence of chloroquine indicated that it acted preferentially on transcription. Differential sensitivity of outgrowing spores and vegetative cells to chloroquine was not due to changes in the specificity of the RNA-polymerase, since RNA-polymerase activity measured in permeabilized cells was not affected differently by the drug. The preferential inhibition of spore outgrowth was not evident at pH 8.0, at which the majority of chloroquine is in a monovalent, more lipophilic, form. In the presence of inhibitors affecting membrane potential, vegetative cells were as sensitive to chloroquine as outgrowing spores. Measurement of [14C]-chloroquine uptake showed that early outgrowing spores accumulated twice as much drug as resistant late outgrowing spores and seven times more than vegetative cells. Treatment of vegetative cells with metabolic inhibitors led them to accumulate chloroquine to the levels found in outgrowing spores. Therefore, the preferential inhibition of outgrowing spores by chloroquine is the result of increased uptake of the drug, reflecting differences in energy metabolism from vegetative cells.

Bacillus subtilis↗
PubMed1978

Viability of spores after repeated freezing and thawing shocks.

Survival of spores of the fungus Rhizopus nigricans after repeated freezing and thawing was investigated. The cooling rate was 10(4) degrees C/min. Dry spores were fully inactive after 32 repeated shocks. About one-half of spores were killed after 8 repetitions. The water content did not change the resistance, swollen spores reacted to shocks much like dry ones. The sensitivity of spores to freezing-thawing shocks increased considerably when the spores changed from the dormant to the active state. Already after a 30 min cultivation of spores in the nutrient medium two freezing and thawings were sufficient for inactivation of 60% spores. After a 90 min cultivation one freezing and one thawing were sufficient to inactivate practically all spores.

Culture Media↗
PubMed2004

A rapid biosensor for viable B. anthracis spores.

A simple membrane-strip-based biosensor assay has been combined with a nucleic acid sequence-based amplification (NASBA) reaction for rapid (4 h) detection of a small number (ten) of viable B. anthracis spores. The biosensor is based on identification of a unique mRNA sequence from one of the anthrax toxin genes, the protective antigen ( pag), encoded on the toxin plasmid, pXO1, and thus provides high specificity toward B. anthracis. Previously, the anthrax toxins activator ( atxA) mRNA had been used in our laboratory for the development of a biosensor for the detection of a single B. anthracis spore within 12 h. Changing the target sequence to the pag mRNA provided the ability to shorten the overall assay time significantly. The vaccine strain of B. anthracis (Sterne strain) was used in all experiments. A 500-microL sample containing as few as ten spores was mixed with 500 microL growth medium and incubated for 30 min for spore germination and mRNA production. Thus, only spores that are viable were detected. Subsequently, RNA was extracted from lysed cells, selectively amplified using NASBA, and rapidly identified by the biosensor. While the biosensor assay requires only 15 min assay time, the overall process takes 4 h for detection of ten viable B. anthracis spores, and is shortened significantly if more spores are present. The biosensor is based on an oligonucleotide sandwich-hybridization assay format. It uses a membrane flow-through system with an immobilized DNA probe that hybridizes with the target sequence. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye sulforhodamine B. The amount of liposomes captured in the detection zone can be read visually or quantified with a hand-held reflectometer. The biosensor can detect as little as 1 fmol target mRNA (1 nmol L(-1)). Specificity analysis revealed no cross-reactivity with 11 organisms tested, among them closely related species such as B. cereus, B. megaterium, B. subtilis, B. thuringiensis, Lactococcus lactis, Lactobacillus plantarum, and Chlostridium butyricum. Also, no false positive signals were obtained from nonviable spores. We suggest that this inexpensive biosensor is a viable option for rapid, on-site analysis providing highly specific data on the presence of viable B. anthracis spores.

Bacillus anthracis↗
PubMed1998

Determination of erythema-effective solar radiation in Japan and Germany with a spore monolayer film optimized for the detection of UVB and UVA--results of a field campaign.

The available physical and biological broad-band radiometers designed to determine erythema-effective radiation do not show any response or over/underestimate the biologically effective radiation to a high extent in the ultraviolet (UV)A spectral region. The data presented in this paper demonstrate that the biological system used in this study is the first one to make possible measurements of erythema-effective radiation in the sun in the UVA and UVB spectral region. These measurements were performed with a spore-film filter system as well as with spectroradiometers. It was demonstrated that this biotechnological method could be used to determine exact values expressed as minimal erythemal dose (MED). The spore-film system was tested in various field campaigns performed in Germany and in Japan. The seasonal daily variation of UV radiation in Germany determined in the period November 1995 to December 1996 using the spore-film filter system in sunny conditions tallied well with model calculations. The daily dose in Germany measured with the spore-film system close to the summer solstice, in sunny conditions (20.45 MED), was approximately 20 times higher than the lowest value measured close to the winter solstice (0.82 MED), a result which was in accordance with model calculations. The data determined with the spore-film filter system in Sapporo and Naha, Japan, fitted to the erythema-weighted data calculated from spectroradiometric measurements (Brewer), even at low solar radiation angles in a solar spectrum with less UVB but significant UVA. The spore-film dosimeter values were about 103 +/- 8% of the integrated dose of the Brewer instrument. The standard deviation of the spore-film measurements obtained in Japan was 12.8%. The responsivity of the spore-film system towards longer wavelengths within the UVA spectrum was tested with the Okasaki Large Spectrograph with monochromatic radiation. At a wavelength of 365 nm--in a spectral region which is dominant in many tanning lamps and with minor importance for solar radiation in summer conditions--the tested spore-film system gave results that were close (112% compared to the calibration dose) to the calibration dose which was used for irradiation.

Bacillus subtilis↗
PubMed2004

Diversity of arbuscular mycorrhizal fungi across a fragmented forest in Panama: insular spore communities differ from mainland communities.

It is now understood that alterations in the species composition of soil organisms can lead to changes in aboveground communities. In this study, we assessed the importance of spatial scale and forest size on changes in arbuscular mycorrhizal fungal (AMF) spore communities by sampling AMF spores in soils of forested mainland and island sites in the vicinity of Gatun Lake, Republic of Panama. We encountered a total of 27 AMF species or morphospecies, with 17, 8, 1 and 1 from the genera Glomus, Acaulospora, Sclerosystis, and Scutellospora, respectively. At small scales (<100 m2), we found little evidence for spatial structuring of AMF communities (decay of Morisita-Horn community similarity with distance). However, at large spatial scales, we found that the AMF spore community of a mainland plot was more similar to other mainland plots several kilometers (>5) away than to nearby island plots (within 0.7 km). Likewise, most island plots were more similar to other island plots regardless of geographic separation. There was no decay in AMF species richness (number of species), or Shannon diversity (number of species and their spore numbers) either with decreasing forest-fragment size, or with decreasing plant species richness. Of the six most common species that composed almost 70% of the total spore volume, spores of Glomus "tsh" and G. clavisporum were more common in soils of mainland plots, while spores of Glomus "small brown" and Acaulospora mellea were more abundant in soils of island plots. None of these common AMF species showed significant associations with soil chemistry or plant diversity. We suggest that the convergence of common species found in AMF spore communities in soils of similar forest sizes was a result of forest fragmentation. Habitat-dependent convergence of AMF spore communities may result in differential survival of tree seedlings regenerating on islands versus mainland.

Analysis of Variance↗