Lyophilization of the spores and vegetative cells of Bacillus brevis var. G-B.--producer of gramicidin S

Viability, antibiotic properties and variation of 4 variants of Bac. brevis var. G.-B. were studied after lyophilization and storage for a year in the lyophilized state. It was shown that the spores and vegetative cells of S and P- variants not synthesizing gramicidin S were somewhat more stable than the spores and cells of R and P+ variants producing the antibiotic. The latter dissociated by 10 per cent towards the cells producing and not producing gramicidin. The developmental rate of the lyophilized vegetative cells was higher than that of the lyophilized spores. Under analogous cultivation conditions they produced higher amounts of the biomass and antibiotic. The lyophilization method described may be recommended for the maintenance of viability and stability of the spores and vegetative cells of Bacillus brevis var. G.-B. producing gramicidin S.     

Impact of wildfire return interval on the ectomycorrhizal resistant propagules communities of a Mediterranean open forest

Ectomycorrhizal (ECM) fungi, in particular their spores and other resistant propagules, play an important role in secondary succession processes that facilitate regeneration after disturbance events. In this study, the effects of high and low wildfire frequencies (respectively short and long fire return intervals) on the resistant propagules communities (RPCs) of a Mediterranean open pine forest were compared. Soil samples were collected in four mountain sites with different fire return intervals and used to test ectomycorrhiza development in two hosts, Pinus pinaster and Quercus suber. RPCs were characterized by direct sequencing of fungal internal transcribed spacer (ITS) regions from individual ECM root tips. Eighteen ECM species were detected in the bioassay. The most frequently found fungi were Cenococcum geophilum, Inocybe jacobi, Thelephora terrestris, Tomentella ellisii on both hosts and Rhizopogon luteolus and R. roseolus on maritime pine. A short fire return interval reduced the species richness of the ECM community found on Q. suber, promoted species like R. roseolus and reduced the abundance of other species (e.g. R. luteolus). The abundance of I. jacobi was positively affected by long fire return interval, but decreased significantly with recurrent fires. These results indicate that changes in fire frequency can alter the structure, composition and diversity of ECM communities, which could compromise the resilience of the ecosystem in highly disturbed areas.     

Base-change mutations induced by various treatments of Bacillus subtilis spores with and without DNA protective small,acid-soluble spore proteins

Previous work has shown that spores of wild-type Bacillus subtilis are more resistant to killing by dry and wet heat, low vacuum lyophilization and hydrogen peroxide than are spores lacking the majority of their DNA protective alpha/beta-type small, acid-soluble spore proteins (SASP) (termed alpha(-)beta(-) spores). These four treatments kill alpha(-)beta(-) spores in large part by DNA damage with accompanying mutagenesis, but only dry heat kills wild-type spores by DNA damage and mutagenesis. DNA sequence analysis of nalidixic acid-resistant (nal(r)) mutants generated by these treatments has now shown that the nal(r) mutations are base changes in the gyrA gene that encodes one subunit of DNA gyrase. Analysis of the DNA sequence of the gyrA gene in a large number of nal(r) mutants also indicates that: (1) base changes induced by hydrogen peroxide and wet heat in alpha(-)beta(-) spores are similar to those in spontaneous nal(r) mutants with only a few notable differences; (2) base changes induced by dry heat in wild-type spores and low vacuum lyophilization of alpha(-)beta(-) spores are similar, and include a high level of a tandem base change seen previously only in spores treated with very high vacuum and (3) base changes induced by lyophilization and dry heat are very different from those in spontaneous mutants in wild-type and alpha(-)beta(-) spores, which exhibit only one significant difference. While the initial DNA damage generated in spores by dry heat, lyophilization or high vacuum is almost certainly different than that generated by hydrogen peroxide or wet heat, the precise nature of the DNA damage remains to be determined.     

Use of acridine yperite in the selection of Act. nodosus, a producer of amphotericin B]

The effect of acridine-yperite (AY) on Act. nodosus LIA 0861 at various physiological stages of development was studied. It was shown that both the spores and the germs were sensitive to AY. The level of the mutagen effect depended on the exposure time and the physiological state of the Act. nodosus cells during the treatment. The treatment of the spore suspension with AY had an insignificant effect on the strain morphological variation, while the number of the morphologically changed colonies markedly decreased when 5-hour germs were subjected to the mutagen effect. A decrease in the average antibiotic activity of Act. nodosus was observed, when its spores were subjected to the treatment with AY, which was associated with a decrease in the number of the plus variants. The treatment of 5-hour germs increased the average antibiotic activity of the culture which correlated with an increase in the number of the plus variants.     

Triggering Germination Represents a Novel Strategy to Enhance Killing of Clostridium difficile Spores

Background

Clostridium difficile is an anaerobic, spore-forming bacterium that is the most common cause of healthcare-associated diarrhea in developed countries. Control of C. difficile is challenging because the spores are resistant to killing by alcohol-based hand hygiene products, antimicrobial soaps, and most disinfectants. Although initiation of germination has been shown to increase susceptibility of spores of other bacterial species to radiation and heat, it was not known if triggering of germination could be a useful strategy to increase susceptibility of C. difficile spores to radiation or other stressors.

Principal Findings

Here, we demonstrated that exposure of dormant C. difficile spores to a germination solution containing amino acids, minerals, and taurocholic acid resulted in initiation of germination in room air. Germination of spores in room air resulted in significantly enhanced killing by ultraviolet-C (UV-C) radiation and heat. On surfaces in hospital rooms, application of germination solution resulted in enhanced eradication of spores by UV-C administered by an automated room decontamination device. Initiation of germination under anaerobic, but not aerobic, conditions resulted in increased susceptibility to killing by ethanol, suggesting that exposure to oxygen might prevent spores from progressing fully to outgrowth. Stimulation of germination also resulted in reduced survival of spores on surfaces in room air, possibly due to increased susceptibility to stressors such as oxygen and desiccation.

Conclusions

Taken together, these data demonstrate that stimulation of germination could represent a novel method to enhance killing of spores by UV-C, and suggest the possible application of this strategy as a means to enhance killing by other agents.     

Application of the real-time PCR for the detection of airborne microbial pathogens in reference to the anthrax spores

To establish the rapid detection method of airborne bacterial spores, we examined Bacillus anthracis spores by real-time PCR. One hundred liters of air were trapped on a filter of an air monitor device. After it was suspended in PBS, spores of B. anthracis were artificially added. The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using anthrax-specific primers. A single cell of B. anthracis was detected by real-time PCR within 1 h. Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR provides a flexible and powerful tool to prevent epidemics.     

Fungal fragments as indoor air biocontaminants

The aerosolization process of fungal propagules of three species (Aspergillus versicolor, Penicillium melinii, and Cladosporium cladosporioides) was studied by using a newly designed and constructed aerosolization chamber. We discovered that fungal fragments are aerosolized simultaneously with spores from contaminated agar and ceiling tile surfaces. Concentration measurements with an optical particle counter showed that the fragments are released in higher numbers (up to 320 times) than the spores. The release of fungal propagules varied depending on the fungal species, the air velocity above the contaminated surface, and the texture and vibration of the contaminated material. In contrast to spores, the release of fragments from smooth surfaces was not affected by air velocity, indicating a different release mechanism. Correlation analysis showed that the number of released fragments cannot be predicted on the basis of the number of spores. Enzyme-linked immunosorbent assays with monoclonal antibodies produced against Aspergillus and Penicillium fungal species showed that fragments and spores share common antigens, which not only confirmed the fungal origin of the fragments but also established their potential biological relevance. The considerable immunological reactivity, the high number, and the small particle size of the fungal fragments may contribute to human health effects that have been detected in buildings with mold problems but had no scientific explanation until now. This study suggests that future fungal spore investigations in buildings with mold problems should include the quantitation of fungal fragments.     

Comparison of Pressure Resistances of Spores of Six Bacillus Strains with Their Heat Resistances

The pressure resistances of the spores of six Bacillus strains were examined at 5 to 10(deg)C and were compared with their heat resistances. The pressure treatments (at 981 MPa for 40 min and at 588 MPa for 120 min) did not inactivate the spores of B. stearothermophilus IAM12043, B. subtilis IAM12118, and B. licheniformis IAM13417. However, these spores had large differences in heat resistance. The spores of B. megaterium IAM1166 were 9.3 times more pressure resistant but 246 times less heat resistant than those of B. stearothermophilus IAM11001. The spores of B. coagulans IAM1194 were activated by the pressure treatments. There was no correlation between these pressure and heat resistances.     

Dispersal of fungal spores from three types of air handling system duct material

Exposure to airborne microorganisms in indoor environments may result in infectious disease or elicit an allergic or irritant response. Air handling system components contaminated by fungi have been implicated in the dispersal of spores into the indoor environment, thereby serving as a route of exposure to occupants. This study was conducted to provide quantitative data on the dispersal of spores from fungal colonies growing on three types of duct material. Galvanized metal, rigid fibrous glass ductboard, and fiberglass duct liner were soiled and contaminated with a known concentration of Penicillium chrysogenum spores. The duct materials were incubated in humidity chambers to provide a matrix of growing, sporulating fungal colonies at a contamination level of 109 colony forming units (CFU) per duct section, consistent for all materials. For each experiment a contaminated duct section was inserted into the air handling system of an experimental room, and the air handling system was operated for three 5-minute cycles with an air flow of 4.2 m3 min–1. The duct air velocity was approximately 2.8 m sec–1. The airborne concentration of culturable P. chrysogenum spores (CFU m–3), total P. chrysogenum spores (spores m–3), and total P. chrysogenum-sized particles (particles m–3) were measured in the room using Andersen single-stage impactor samplers, Burkard slide impactor samplers, and an aerodynamic particle sizer, respectively. The highest airborne concentrations (104 CFU m–3; 105 spores m–3; 104 particles m–3) were measured during the first operating cycle of the air handling system for all duct materials with decreasing airborne concentrations measured during the second and third cycles. There was no significant difference in spore dispersal from the three contaminated duct materials. These data demonstrate the potential exposure for building occupants to high concentrations of spores dispersed from fungal colonies on air handling system duct materials during normal operation of the system.     

Emission rate of charged spores in basidiomycetous fungi and the relaxation time of their electric charges

Basidiospores are one of the main components of coarse fraction of atmospheric aerosol. Majority of them, the ballistospores of 20,000 species of Basidiomycotina, carry electrostatic charges when getting airborne. To study the polarity and magnitude of primary charges and the hymenial emission rate of charged spores, 128 spore samples of 31 species of Agaricomycetes were collected in natural conditions. A portable device was placed under the fruiting body and the freely falling charged spores were extracted from the air by a horizontal homogeneous electrostatic field. The charge polarity distribution was the same in all intraspecies spore samples; it was unipolar-positive, unipolar-negative, or bipolar, depending on the species. The mean spore charge magnitude was 21–981 e, and it was not related to the emission rate of charged spores. The hymenial emission rate was fluctuating, and the maximum value was 715 charged spores cm^?2 s^?1. To estimate the territorial emission rate of charged spores, area of the hymenial surface per hectare of forest was calculated for three species and the maximum values were 11 m² ha^?1 and 8.6 × 10⁷ charged spores ha^?1 s^?1. Calculations showed that a spore charge diminished sevenfold within 47 min. Ecologists, health and agricultural scientists could be interested in this information. It could be useful by investigating the role of microorganisms in meteorological phenomena and in atmospheric processes in general.     

The preservation of infective spores of Octosporea muscaedomesticae in Phormia regina,of Nosema algerae in Anopheles stephensi,and of Nosema whitei in Tribolium castaneum by lyophilization

Infective spores of three species of microsporidia were subjected to the lyophilization process by employing varying media as cryoprotectants. The infectivity of the lyophilized spores was then tested against a standard fresh spore preparation in the appropriate host insect. Spores of Octosporea muscaedomesticae served as an experimental model and were rendered noninfective in host Phormia regina (Calliphoridae: Diptera) after lyophilization with the following cryoprotective agents: skim milk (12%), ascorbic acid (5%) combined with thiourea (5%), glycerol (10%), mesoinositol (5%), and equine serum. Spores of O. muscaedomesticae lyophilized or vacuum-dried in 50% sucrose as well as in the hosts' tissues remained highly infective for as long as 2 years at a dose of 10⁶ spores/fly and a trial length of 12 days. At a dose of 5 × 10⁴ spores/fly there was a slight decrease in infectivity of the spores which had been lyophilized in the host's abdomen after a 2-year storage period compared with that of fresh, nonlyophilized spores. Naked spores of Nosema algerae suspended in 50% sucrose and lyophilized produced infection in 50% of the host population of Anopheles stephensi (Culicidae: Diptera) compared with 70% infection produced by fresh non-lyophilized spores. Spores of Nosema whitei lyophilized within its host larva Tribolium castaneum (Tenebrionidae: Coleoptera) remained 100% infective at a dose of 5 × 10⁵ spores/gram diet. It is concluded that an aqueous solution of 50% sucrose and/or the host's tissues are excellent protectants for the cryogenic or vacuum-drying process of the above-named spores, and their protective function may apply also to other microsporidian species.     

Bees Scavenge Airborne Bacteria

Abstract An air conditioned wind tunnel system was designed, fabricated, and tested to determine whether tethered bees scavenge microbeads or Bacillus subtilis var. niger spores from aerosols. Tests showed that microbeads and spores were scavenged by bumblebees and honeybees, respectively. Five independent variables and their interactions were used in a stepwise multiple regression. Two of them, the cube root of the electrostatic charge on the honeybee and the dose of the spore aerosol, accounted for most of the statistically significant fit to the model's two dependent variables: the percentage of the dose adsorbed by honeybees and the number of spores adsorbed by the same bees. Both dependent variables increased directly so that an increase in electrostatic charge on the bee (i.e., cube root 32 pC) resulted in an increase (i.e., approximately 1%) in the spore dose adsorbed and the number of spores adsorbed by the bees. It was theorized that the spores were in an adsorption/desorption equilibrium that responded to the concentration ``pressure' of the spore aerosol. Further, the charge on the bee affected the adsorption force on the bee's surface, as well as increasing the effective aerosol volume accessible for the bee's scavenging. In short, relating these findings to bees scavenging bacteria from the ambient atmosphere, it appears that the spore exposure (where exposure means the product of the ambient concentration, the time the bee is exposed, and air volume through which the bee flies) controls the number of spores adsorbed by a bee, and the static charge on the bee controls the adsorption/desorption equilibrium and presumably the scavenging volume. Received: 22 November 1999; Accepted: 21 January 2000; Online Publication: 29 May 2000     

Tracking mycorrhizas and extraradical mycelium of the edible fungus <Emphasis Type="Italic">Lactarius deliciosus</Emphasis> under field competition with <Emphasis Type="Italic">Rhizopogon</Emphasis> spp

The objective of this study is to evaluate the field persistence of the edible ectomycorrhizal fungus Lactarius deliciosus in competition with two ubiquitous soil fungi. Couples of plants inoculated with either L. deliciosus, Rhizopogon roseolus, or R. luteolus were transplanted, 10 cm apart, in two different sites at the following combinations: L. deliciosus-R. roseolus, L. deliciosus-R. luteolus, L. deliciosus-control (non-inoculated), control-R. roseolus, control-R. luteolus, and control-control. Eight months after transplantation, root colonization and extraradical soil mycelium for each fungal species were quantified. For mycelium quantification, soil cores equidistant to the two plants in each couple were taken, and total deoxyribonucleic acid (DNA) was extracted. Real-time polymerase chain reaction analysis was performed using specific primers and TaqMan Minor groove binding (MGB) probes designed in the ribosomal DNA internal transcribed spacer region of each fungal species. Field site significantly influenced persistence of both mycorrhizas and extraradical mycelium of L. deliciosus. Extraradical mycelium quantity was positively correlated with the final percentage of ectomycorrhizas for the three fungal species. Different competitive pressure between the two Rhizopogon species on L. deliciosus persistence was observed, with R. luteolus having no effect on L. deliciosus survival. Negative correlation between the final percentage of mycorrhizas of L. deliciosus and R. roseolus was observed. However, no relationship was determined between extraradical mycelia of both fungal species. The results obtained suggest that competition between L. deliciosus and R. roseolus takes place in the root system, for ectomycorrhiza formation in available roots, rather than in the extraradical phase.     

Preservation of aerial conidia and biomasses from entomopathogenic fungi Beauveria brongniartii and Metarhizium anisopliae during lyophilization

In this study, we assessed the stability provided by different formulations to aerial conidia or biomasses (conidia, blastospores, and mycelia) of Beauveria brongniartii and Metarhizium anisopliae subjected to lyophilization. First, the impact of the freezing and drying processes on spore survival was evaluated. Whereas unprotected B. brongniartii spores showed high cryosensitivity, those of M. anisopliae were markedly harmed by the drying process. Then, the protective efficiency of 14 excipients was systematically evaluated and optimized regarding required concentrations. Fructose, glucose, and saccharose significantly enhanced viabilities for B. brongniartii and M. anisopliae spores following lyophilization, especially as a result of their cryoprotective effects. In addition, the effect of various bulking agents on spore survival was studied and dextran 4 was selected to enhance the physical properties of the lyophilized products. The combination of fructose and dextran 4 was further applied to prepare lyophilized biomasses of both fungi. In comparison to freshly harvested biomasses, the lyophilized products showed similar growth rates and a comparable production of virulent secondary metabolites such as destruxin A, destruxin B, or oosporein, suggesting their applicability as biological control agents.     

Fungal Fragments as Indoor Air Biocontaminants

The aerosolization process of fungal propagules of three species (Aspergillus versicolor, Penicillium melinii, and Cladosporium cladosporioides) was studied by using a newly designed and constructed aerosolization chamber. We discovered that fungal fragments are aerosolized simultaneously with spores from contaminated agar and ceiling tile surfaces. Concentration measurements with an optical particle counter showed that the fragments are released in higher numbers (up to 320 times) than the spores. The release of fungal propagules varied depending on the fungal species, the air velocity above the contaminated surface, and the texture and vibration of the contaminated material. In contrast to spores, the release of fragments from smooth surfaces was not affected by air velocity, indicating a different release mechanism. Correlation analysis showed that the number of released fragments cannot be predicted on the basis of the number of spores. Enzyme-linked immunosorbent assays with monoclonal antibodies produced against Aspergillus and Penicillium fungal species showed that fragments and spores share common antigens, which not only confirmed the fungal origin of the fragments but also established their potential biological relevance. The considerable immunological reactivity, the high number, and the small particle size of the fungal fragments may contribute to human health effects that have been detected in buildings with mold problems but had no scientific explanation until now. This study suggests that future fungal spore investigations in buildings with mold problems should include the quantitation of fungal fragments.     

Photocontrol of spore germination in the fern Thelypteris kunthii

The light requirement for germination in spores of the fern Thelypteris kunthii (Desv.) Morton was fully satisfied by a long period of continuous red light or partially by intermittent, short periods of red light. Red light-potentiated spore germination was inhibited by brief far-red light irradiation, indicating phytochrome involvement. Repeated exposure of spores to prolonged red and short far-red irradiations, or exposure of red-potentiated spores to far-red light after an extended period in darkness, led to their escape from inhibition of germination by far-red light. Prolonged irradiation of spores with blue light before or after red light treatment partially antagonized the effect of red light.     

Comparison of airborne spore concentrations and fungal allergen content

The exposure to spores causing health effects is usually assessed by determining the concentration of viable spores per cubic meter of air (CFU/m³).Since allergens might also be present in dead spores or smaller particles, the objective of this study was to investigate the correlation between the viable spores of Alternaria and Cladosporium at different indoor and outdoor sites and the corresponding allergen concentration detected with a specially developed ELISA (Enzyme Linked Immunosorbent Assay). In outdoor air, the results show a strong correlation between the different sampling techniques applied for viable spores (Slit-Sampler and Multistage Liquid Impinger) and between the viable spores and the allergen concentrations detected in the liquid samples of the impingers. Indoors, the number of viable spores and the allergen concentration do not correlate and the allergen load is underestimated if colony counting methods are used. This revised version was published online in June 2006 with corrections to the Cover Date.     

Whirling disease: host specificity and interaction between the actinosporean stage of Myxobolus cerebralis and rainbow trout Oncorhynchus mykiss

Scanning electron microscopic studies were conducted on rainbow trout Oncorhynchus mykiss in the first 60 min after their exposure to the triactinomyxon spores of Myxobolus cerebralis. The results demonstrated that as early as 1 min post exposure the whole process, from the attachment of the triactinomyxon spores to the complete penetration of their sporoplasm germs, had occurred. The triactinomyxon spores sought out the secretory openings of mucous cells of the epidermis, the respiratory epithelium and the buccal cavity of trout and used them as portals of entry. Exposure experiments of the triactinomyxon spores of M. cerebralis to non-salmonid fish, such as goldfish Carassius auratus, carp Cyprinus carpio, nose Chondrostoma nasus, medaka Oryzias latipes, guppy Poecilia reticulata and also the amphibian tadpole Rana pipiens as well as to rainbow trout fry indicated a specificity for salmonids. Attempts to activate the triactinomyxon spores by exposure to mucus prepared from cyprinid and salmonid fish showed no significant differences from those conducted in tap water. The results suggest that the simultaneous presence of both mechano- and chemotactic stimuli was required for finding the salmonid fish host.     

Detection of arbuscular mycorrhizal fungal spores in the air across different biomes and ecoregions

Aerial dispersal of fungal spores is common, but the role of wind and air movement in dispersal of spores of arbuscular mycorrhizal (AM) fungi is largely unknown. Several studies have examined the possibility of AM fungal spores being moved by wind vectors without observing spores taken from the air environment. For the first time this study observed the presence of AM fungal spores in the air. The frequency of AM fungal spores in the air was determined in six North American biomes composed of 18 ecoregions. Multiple samples were taken from both the air and the soil at each location. AM fungal spores were found in high abundance in the soil (hundreds of spores per gram of soil), however, they were rarely found in the air (most samples contained no AM fungal spores). Furthermore, only the Glomus morphotype was found in the air, whereas spores in the soil were taxomomically more diverse (Glomus, Acaulospora, Gigaspora, Scutellospora morphotypes were observed). The proportion of Glomus spores in the air relative to Glomus spores in the soil was highest in more arid systems, indicating that AM fungi may be more likely to be dispersed in the air in such systems. Nonetheless, the results indicate that the air is not likely a dominant mode of dispersal for AM fungi.     

Blue Light Interference in the Phytochrome-controlled Germination of the Spores of Cheilanthes farinosa

Short exposure of the spores of Cheilanthes farinosa to low intensity red light promotes their germination, which is not reversed by a subsequent exposure to far red light. Germination is, however, inhibited by blue light administered before or after red light. Inhibition of germination by blue light is annulled by exposure to a higher intensity of red light, and germination of the repromoted spores is inhibited by far red light. Mutual photoreversibility of germination is also observed in repromoted spores irradiated successively with far red and red light. Although germination appears to be basically under phytochrome control, it is postulated that the presence of a blue light-absorbing pigment interferes with phytochrome transformations in the spores.