Aerosol and Surface Deposition Characteristics of Two Surrogates for Bacillus anthracis Spores

PubMed Central

Stapleton, Helen L.

2016-01-01

ABSTRACT Spores of an acrystalliferous derivative of Bacillus thuringiensis subsp. kurstaki, termed Btcry−, are morphologically, aerodynamically, and structurally indistinguishable from Bacillus anthracis spores. Btcry− spores were dispersed in a large, open-ended barn together with spores of Bacillus atrophaeus subsp. globigii, a historically used surrogate for Bacillus anthracis. Spore suspensions (2 × 1012 CFU each of B. atrophaeus subsp. globigii and Btcry−) were aerosolized in each of five spray events using a backpack misting device incorporating an air blower; a wind of 4.9 to 7.6 m s−1 was also flowing through the barn in the same direction. Filter air samplers were situated throughout the barn to assess the aerosol density of the spores during each release. Trays filled with a surfactant in aqueous buffer were placed on the floor near the filter samplers to assess spore deposition. Spores were also recovered from arrays of solid surfaces (concrete, aluminum, and plywood) that had been laid on the floor and set up as a wall at the end of the barn. B. atrophaeus subsp. globigii spores were found to remain airborne for significantly longer periods, and to be deposited on horizontal surfaces at lower densities, than Btcry− spores, particularly near the spray source. There was a 6-fold-higher deposition of Btcry− spores than of B. atrophaeus subsp. globigii spores on vertical surfaces relative to the surrounding airborne density. This work is relevant for selecting the best B. anthracis surrogate for the prediction of human exposure, hazard assessment, and hazard management following a malicious release of B. anthracis. IMPORTANCE There is concern that pathogenic bacteria could be maliciously disseminated in the air to cause human infection and disruption of normal life. The threat from spore-forming organisms, such as the causative agent of anthrax, is particularly serious. In order to assess the extent of this risk, it is important to have a surrogate organism that can be used to replicate the dispersal characteristics of the threat agent accurately. This work compares the aerosol dispersal and deposition behaviors of the surrogates Btcry− and B. atrophaeus subsp. globigii. Btcry− spores remained in the air for a shorter time, and were markedly more likely to adhere to vertical surfaces, than B. atrophaeus subsp. globigii spores. PMID:27613681

A Streptomyces-specific member of the metallophosphatase superfamily contributes to spore dormancy and interaction with Aspergillus proliferans.

PubMed

Lamp, Jessica; Weber, Maren; Cingöz, Gökhan; Ortiz de Orué Lucana, Darío; Schrempf, Hildgund

2013-05-01

We have identified, cloned and characterized a formerly unknown protein from Streptomyces lividans spores. The deduced protein belongs to a novel member of the metallophosphatase superfamily and contains a phosphatase domain and predicted binding sites for divalent ions. Very close relatives are encoded in the genomic DNA of many different Streptomyces species. As the deduced related homologues diverge from other known phosphatase types, we named the protein MptS (metallophosphatase type from Streptomyces). Comparative physiological and biochemical investigations and analyses by fluorescence microscopy of the progenitor strain, designed mutants carrying either a disruption of the mptS gene or the reintroduced gene as fusion with histidine codons or the egfp gene led to the following results: (i) the mptS gene is transcribed in the course of aerial mycelia formation. (ii) The MptS protein is produced during the late stages of growth, (iii) accumulates within spores, (iv) functions as an active enzyme that releases inorganic phosphate from an artificial model substrate, (v) is required for spore dormancy and (vi) MptS supports the interaction amongst Streptomyces lividans spores with conidia of the fungus Aspergillus proliferans. We discuss the possible role(s) of MptS-dependent enzymatic activity and the implications for spore biology. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

A vacuolar membrane protein Avt7p is involved in transport of amino acid and spore formation in Saccharomyces cerevisiae.

PubMed

Tone, Junichi; Yamanaka, Atsushi; Manabe, Kunio; Murao, Nami; Kawano-Kawada, Miyuki; Sekito, Takayuki; Kakinuma, Yoshimi

2015-01-01

Active transport systems for various amino acids operate in the vacuolar membrane of Saccharomyces cerevisiae. The gene families for vacuolar amino acid transporters were identified by reverse genetics experiments. In the AVT transporter family, Avt1p works for active uptake of amino acid into vacuole, and Avt3p, Avt4p, and Avt6p for active extrusion of amino acid from vacuole to cytosol. Here, we found green fluorescent protein-tagged Avt7p, an unidentified member of the AVT family, localized to the vacuolar membrane of S. cerevisiae. Disruption of the AVT7 gene enhanced both vacuolar contents of several amino acids and uptake activities of glutamine and proline by vacuolar membrane vesicles. Efficiency of spore formation was impaired by the disruption of the AVT7 gene, suggesting the physiological importance of Avt7p-dependent efflux of amino acid from vacuoles under nutrient-poor condition.

Fatty Acid Oxidation Is Required for Myxococcus xanthus Development.

PubMed

Bullock, Hannah A; Shen, Huifeng; Boynton, Tye O; Shimkets, Lawrence J

2018-05-15

Myxococcus xanthus cells produce lipid bodies containing triacylglycerides during fruiting body development. Fatty acid β-oxidation is the most energy-efficient pathway for lipid body catabolism. In this study, we used mutants in fadJ (MXAN_5371 and MXAN_6987) and fadI (MXAN_5372) homologs to examine whether β-oxidation serves an essential developmental function. These mutants contained more lipid bodies than the wild-type strain DK1622 and 2-fold more flavin adenine dinucleotide (FAD), consistent with the reduced consumption of fatty acids by β-oxidation. The β-oxidation pathway mutants exhibited differences in fruiting body morphogenesis and produced spores with thinner coats and a greater susceptibility to thermal stress and UV radiation. The MXAN_5372/5371 operon is upregulated in sporulating cells, and its expression could not be detected in csgA , fruA , or mrpC mutants. Lipid bodies were found to persist in mature spores of DK1622 and wild strain DK851, suggesting that the roles of lipid bodies and β-oxidation may extend to spore germination. IMPORTANCE Lipid bodies act as a reserve of triacylglycerides for use when other sources of carbon and energy become scarce. β-Oxidation is essential for the efficient metabolism of fatty acids associated with triacylglycerides. Indeed, the disruption of genes in this pathway has been associated with severe disorders in animals and plants. Myxococcus xanthus , a model organism for the study of development, is ideal for investigating the complex effects of altered lipid metabolism on cell physiology. Here, we show that β-oxidation is used to consume fatty acids associated with lipid bodies and that the disruption of the β-oxidation pathway is detrimental to multicellular morphogenesis and spore formation. Copyright © 2018 American Society for Microbiology.

Next-Generation Bacillus anthracis Live Attenuated Spore Vaccine Based on the htrA- (High Temperature Requirement A) Sterne Strain

PubMed Central

Chitlaru, Theodor; Israeli, Ma’ayan; Bar-Haim, Erez; Elia, Uri; Rotem, Shahar; Ehrlich, Sharon; Cohen, Ofer; Shafferman, Avigdor

2016-01-01

Anthrax is a lethal disease caused by the gram-positive spore-producing bacterium Bacillus anthracis. Live attenuated vaccines, such as the nonencapsulated Sterne strain, do not meet the safety standards mandated for human use in the Western world and are approved for veterinary purposes only. Here we demonstrate that disrupting the htrA gene, encoding the chaperone/protease HtrA (High Temperature Requirement A), in the virulent Bacillus anthracis Vollum strain results in significant virulence attenuation in guinea pigs, rabbits and mice, underlying the universality of the attenuated phenotype associated with htrA knockout. Accordingly, htrA disruption was implemented for the development of a Sterne-derived safe live vaccine compatible with human use. The novel B. anthracis SterneΔhtrA strain secretes functional anthrax toxins but is 10–104-fold less virulent than the Sterne vaccine strain depending on animal model (mice, guinea pigs, or rabbits). In spite of this attenuation, double or even single immunization with SterneΔhtrA spores elicits immune responses which target toxaemia and bacteremia resulting in protection from subcutaneous or respiratory lethal challenge with a virulent strain in guinea pigs and rabbits. The efficacy of the immune-protective response in guinea pigs was maintained for at least 50 weeks after a single immunization. PMID:26732659

Seminal fluid of honeybees contains multiple mechanisms to combat infections of the sexually transmitted pathogen Nosema apis.

PubMed

Peng, Yan; Grassl, Julia; Millar, A Harvey; Baer, Boris

2016-01-27

The societies of ants, bees and wasps are genetically closed systems where queens only mate during a brief mating episode prior to their eusocial life and males therefore provide queens with a lifetime supply of high-quality sperm. These ejaculates also contain a number of defence proteins that have been detected in the seminal fluid but their function and efficiency have never been investigated in great detail. Here, we used the honeybee Apis mellifera and quantified whether seminal fluid is able to combat infections of the fungal pathogen Nosema apis, a widespread honeybee parasite that is also sexually transmitted. We provide the first empirical evidence that seminal fluid has a remarkable antimicrobial activity against N. apis spores and that antimicrobial seminal fluid components kill spores in multiple ways. The protein fraction of seminal fluid induces extracellular spore germination, which disrupts the life cycle of N. apis, whereas the non-protein fraction of seminal fluid induces a direct viability loss of intact spores. We conclude that males provide their ejaculates with efficient antimicrobial molecules that are able to kill N. apis spores and thereby reduce the risk of disease transmission during mating. Our findings could be of broader significance to master honeybee diseases in managed honeybee stock in the future. © 2016 The Author(s).

Molecular characterization and histopathology of Myxobolus koi infecting the gills of a koi, Cyprinus carpio, with an amended morphological description of the agent.

PubMed

Camus, Alvin C; Griffin, Matt J

2010-02-01

A Myxobolus sp., morphologically resembling M. toyamai, M. longisporus, and M. koi, was isolated from the gills of a koi, Cyprinus carpio, that died in an ornamental pond. Large plasmodia were localized within lamellae, causing severe disruption of the normal branchial architecture, sufficient to compromise respiration. Although the case isolate shared several features with the aforementioned species, several key characteristics were most compatible with M. koi. In valvular view, spores were elongate and pyriform with a rounded posterior, 15.4 (14.5-16.5) microm long and 8.3 (7.1-9.0) microm wide. Polar capsules were pyriform and elongate, 10.1 (9.0-10.9) microm long and 3.1 (2.5-3.5) microm wide. Polar filaments were coiled perpendicular to the long axis of the spore making 10 turns (9-11). A BLAST search using a generated 18S SSU rDNA sequence resulted in no direct matches. Phylogenetic analysis of the sequence indicates a close relationship to M. longisporus and M. toyamai from C. carpio, within the clade of Myxobolus species infecting gills of cyprinid fishes (89% or greater bootstrap support by maximum parsimony and minimum evolution distance analysis). Although published descriptions are inconsistent, morphometric similarities to this isolate suggest a morphotype of M. koi. The description of M. koi is supplemented here with new data on pathogenicity and spore morphology and is characterized at the molecular level using 18S small subunit rDNA sequence data.

A Review of the Disruptive Potential of Botulinum Neurotoxins as Chemical Warfare Agents

DTIC Science & Technology

2011-10-01

canned; fermented , uncooked; or improperly cooked dishes); 2 • Ingestion of spores, which in turn colonize the small intestine (occurs in...should also be noted that some food products may be more amenable to contamination with active BoNT than milk-based dairy products, although for

Diverse microbial species survive high ammonia concentrations

NASA Astrophysics Data System (ADS)

Kelly, Laura C.; Cockell, Charles S.; Summers, Stephen

2012-04-01

Planetary protection regulations are in place to control the contamination of planets and moons with terrestrial micro-organisms in order to avoid jeopardizing future scientific investigations relating to the search for life. One environmental chemical factor of relevance in extraterrestrial environments, specifically in the moons of the outer solar system, is ammonia (NH3). Ammonia is known to be highly toxic to micro-organisms and may disrupt proton motive force, interfere with cellular redox reactions or cause an increase of cell pH. To test the survival potential of terrestrial micro-organisms exposed to such cold, ammonia-rich environments, and to judge whether current planetary protection regulations are sufficient, soil samples were exposed to concentrations of NH3 from 5 to 35% (v/v) at -80°C and room temperature for periods up to 11 months. Following exposure to 35% NH3, diverse spore-forming taxa survived, including representatives of the Firmicutes (Bacillus, Sporosarcina, Viridibacillus, Paenibacillus, Staphylococcus and Brevibacillus) and Actinobacteria (Streptomyces). Non-spore forming organisms also survived, including Proteobacteria (Pseudomonas) and Actinobacteria (Arthrobacter) that are known to have environmentally resistant resting states. Clostridium spp. were isolated from the exposed soil under anaerobic culture. High NH3 was shown to cause a reduction in viability of spores over time, but spore morphology was not visibly altered. In addition to its implications for planetary protection, these data show that a large number of bacteria, potentially including spore-forming pathogens, but also environmentally resistant non-spore-formers, can survive high ammonia concentrations.

Development of size-selective sampling of Bacillus anthracis surrogate spores from simulated building air intake mixtures for analysis via laser-induced breakdown spectroscopy.

PubMed

Gibb-Snyder, Emily; Gullett, Brian; Ryan, Shawn; Oudejans, Lukas; Touati, Abderrahmane

2006-08-01

Size-selective sampling of Bacillus anthracis surrogate spores from realistic, common aerosol mixtures was developed for analysis by laser-induced breakdown spectroscopy (LIBS). A two-stage impactor was found to be the preferential sampling technique for LIBS analysis because it was able to concentrate the spores in the mixtures while decreasing the collection of potentially interfering aerosols. Three common spore/aerosol scenarios were evaluated, diesel truck exhaust (to simulate a truck running outside of a building air intake), urban outdoor aerosol (to simulate common building air), and finally a protein aerosol (to simulate either an agent mixture (ricin/anthrax) or a contaminated anthrax sample). Two statistical methods, linear correlation and principal component analysis, were assessed for differentiation of surrogate spore spectra from other common aerosols. Criteria for determining percentages of false positives and false negatives via correlation analysis were evaluated. A single laser shot analysis of approximately 4 percent of the spores in a mixture of 0.75 m(3) urban outdoor air doped with approximately 1.1 x 10(5) spores resulted in a 0.04 proportion of false negatives. For that same sample volume of urban air without spores, the proportion of false positives was 0.08.

A Bacillus subtilis malate dehydrogenase gene.

PubMed Central

Jin, S; De Jesús-Berríos, M; Sonenshein, A L

1996-01-01

A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase. Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation. In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation. PMID:8550482

Characterizing Aeroallergens by Infrared Spectroscopy of Fungal Spores and Pollen

PubMed Central

Zimmermann, Boris; Tkalčec, Zdenko; Mešić, Armin; Kohler, Achim

2015-01-01

Background Fungal spores and plant pollen cause respiratory diseases in susceptible individuals, such as asthma, allergic rhinitis and hypersensitivity pneumonitis. Aeroallergen monitoring networks are an important part of treatment strategies, but unfortunately traditional analysis is time consuming and expensive. We have explored the use of infrared spectroscopy of pollen and spores for an inexpensive and rapid characterization of aeroallergens. Methodology The study is based on measurement of spore and pollen samples by single reflectance attenuated total reflectance Fourier transform infrared spectroscopy (SR-ATR FTIR). The experimental set includes 71 spore (Basidiomycota) and 121 pollen (Pinales, Fagales and Poales) samples. Along with fresh basidiospores, the study has been conducted on the archived samples collected within the last 50 years. Results The spectroscopic-based methodology enables clear spectral differentiation between pollen and spores, as well as the separation of confamiliar and congeneric species. In addition, the analysis of the scattering signals inherent in the infrared spectra indicates that the FTIR methodology offers indirect estimation of morphology of pollen and spores. The analysis of fresh and archived spores shows that chemical composition of spores is well preserved even after decades of storage, including the characteristic taxonomy-related signals. Therefore, biochemical analysis of fungal spores by FTIR could provide economical, reliable and timely methodologies for improving fungal taxonomy, as well as for fungal identification and monitoring. This proof of principle study shows the potential for using FTIR as a rapid tool in aeroallergen studies. In addition, the presented method is ready to be immediately implemented in biological and ecological studies for direct measurement of pollen and spores from flowers and sporocarps. PMID:25867755

Calcineurin orchestrates dimorphic transitions, antifungal drug responses and host-pathogen interactions of the pathogenic mucoralean fungus Mucor circinelloides.

PubMed

Lee, Soo Chan; Li, Alicia; Calo, Silvia; Inoue, Makoto; Tonthat, Nam K; Bain, Judith M; Louw, Johanna; Shinohara, Mari L; Erwig, Lars P; Schumacher, Maria A; Ko, Dennis C; Heitman, Joseph

2015-09-01

Calcineurin plays essential roles in virulence and growth of pathogenic fungi and is a target of the natural products FK506 and Cyclosporine A. In the pathogenic mucoralean fungus Mucor circinelloides, calcineurin mutation or inhibition confers a yeast-locked phenotype indicating that calcineurin governs the dimorphic transition. Genetic analysis in this study reveals that two calcineurin A catalytic subunits (out of three) are functionally diverged. Homology modeling illustrates modes of resistance resulting from amino substitutions in the interface between each calcineurin subunit and the inhibitory drugs. In addition, we show how the dimorphic transition orchestrated by calcineurin programs different outcomes during host-pathogen interactions. For example, when macrophages phagocytose Mucor yeast, subsequent phagosomal maturation occurs, indicating host cells respond appropriately to control the pathogen. On the other hand, upon phagocytosis of spores, macrophages fail to form mature phagosomes. Cytokine production from immune cells differs following exposure to yeast versus spores (which germinate into hyphae). Thus, the morphogenic transition can be targeted as an efficient treatment option against Mucor infection. In addition, genetic analysis (including gene disruption and mutational studies) further strengthens the understanding of calcineurin and provides a foundation to develop antifungal agents targeting calcineurin to deploy against Mucor and other pathogenic fungi. © 2015 John Wiley & Sons Ltd.

A Novel Regulator Controls Clostridium difficile Sporulation, Motility and Toxin Production

PubMed Central

Edwards, Adrianne N.; Tamayo, Rita; McBride, Shonna M.

2016-01-01

SUMMARY Clostridium difficile, is an anaerobic pathogen that forms spores which promote survival in the environment and transmission to new hosts. The regulatory pathways by which C. difficile initiates spore formation are poorly understood. We identified two factors with limited similarity to the Rap sporulation proteins of other spore-forming bacteria. In this study, we show that disruption of the gene CD3668 reduces sporulation and increases toxin production and motility. This mutant was more virulent and exhibited increased toxin gene expression in the hamster model of infection. Based on these phenotypes, we have renamed this locus rstA, for regulator of sporulation and toxins. Our data demonstrate that RstA is a bifunctional protein that upregulates sporulation through an unidentified pathway and represses motility and toxin production by influencing sigD transcription. Conserved RstA orthologs are present in other pathogenic and industrial Clostridium species and may represent a key regulatory protein controlling clostridial sporulation. PMID:26915493

A novel regulator controls Clostridium difficile sporulation, motility and toxin production.

PubMed

Edwards, Adrianne N; Tamayo, Rita; McBride, Shonna M

2016-06-01

Clostridium difficile is an anaerobic pathogen that forms spores which promote survival in the environment and transmission to new hosts. The regulatory pathways by which C. difficile initiates spore formation are poorly understood. We identified two factors with limited similarity to the Rap sporulation proteins of other spore-forming bacteria. In this study, we show that disruption of the gene CD3668 reduces sporulation and increases toxin production and motility. This mutant was more virulent and exhibited increased toxin gene expression in the hamster model of infection. Based on these phenotypes, we have renamed this locus rstA, for regulator of sporulation and toxins. Our data demonstrate that RstA is a bifunctional protein that upregulates sporulation through an unidentified pathway and represses motility and toxin production by influencing sigD transcription. Conserved RstA orthologs are present in other pathogenic and industrial Clostridium species and may represent a key regulatory protein controlling clostridial sporulation. © 2016 John Wiley & Sons Ltd.

Involvement of Clostridium botulinum ATCC 3502 Sigma Factor K in Early-Stage Sporulation

PubMed Central

Kirk, David G.; Dahlsten, Elias; Zhang, Zhen; Korkeala, Hannu

2012-01-01

A key survival mechanism of Clostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organism Bacillus subtilis, nothing is known about these mechanisms in C. botulinum. Using the ClosTron gene-knockout tool, sigK, encoding late-stage (stage IV) sporulation sigma factor K in B. subtilis, was disrupted in C. botulinum ATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast, sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis of sigK in the parent strain revealed expression at the late log growth phase in the parent strain. Analysis of spo0A, encoding the sporulation master switch, in the sigK mutant and the parent showed significantly reduced relative levels of spo0A expression in the sigK mutant compared to the parent strain. Similarly, sigF showed significantly lower relative transcription levels in the sigK mutant than the parent strain, suggesting that the sporulation pathway was blocked in the sigK mutant at an early stage. We conclude that σK is essential for early-stage sporulation in C. botulinum ATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organism B. subtilis. Understanding the sporulation mechanism of C. botulinum provides keys to control the public health risks that the spores of this dangerous pathogen cause through foods. PMID:22544236

Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation.

PubMed

Kirk, David G; Dahlsten, Elias; Zhang, Zhen; Korkeala, Hannu; Lindström, Miia

2012-07-01

A key survival mechanism of Clostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organism Bacillus subtilis, nothing is known about these mechanisms in C. botulinum. Using the ClosTron gene-knockout tool, sigK, encoding late-stage (stage IV) sporulation sigma factor K in B. subtilis, was disrupted in C. botulinum ATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast, sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis of sigK in the parent strain revealed expression at the late log growth phase in the parent strain. Analysis of spo0A, encoding the sporulation master switch, in the sigK mutant and the parent showed significantly reduced relative levels of spo0A expression in the sigK mutant compared to the parent strain. Similarly, sigF showed significantly lower relative transcription levels in the sigK mutant than the parent strain, suggesting that the sporulation pathway was blocked in the sigK mutant at an early stage. We conclude that σ(K) is essential for early-stage sporulation in C. botulinum ATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organism B. subtilis. Understanding the sporulation mechanism of C. botulinum provides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.

Schizosaccharomyces pombe possesses an unusual and a conventional hexokinase: biochemical and molecular characterization of both hexokinases.

PubMed

Petit, T; Blázquez, M A; Gancedo, C

1996-01-08

Two hexokinases were characterized in Schizosaccharomyces pombe: hexokinase 1, with a low phosphorylation coefficient on glucose (Km 8.5 mM) and hexokinase 2, a kinetically conventional hexokinase. Genes hxk1+ and hxk2+ encoding these enzymes were cloned and sequenced. Disruption of hxk1+ had no effect on growth but disruption of hxk2+ doubled the generation time in glucose. Spores carrying the double disruption hxk1+ hxk2+ did not grow on glucose or fructose after one week. Expression of hxk1+ increased strongly during growth in fructose or glycerol. Expression of hxk2+ was highest during growth in glycerol. A NADP-dependent glucose dehydrogenase was detected, but not a glucokinase.

Inactivation of chemical and heat-resistant spores of Bacillus and Geobacillus by nitrogen cold atmospheric plasma evokes distinct changes in morphology and integrity of spores.

PubMed

van Bokhorst-van de Veen, Hermien; Xie, Houyu; Esveld, Erik; Abee, Tjakko; Mastwijk, Hennie; Nierop Groot, Masja

2015-02-01

Bacterial spores are resistant to severe conditions and form a challenge to eradicate from food or food packaging material. Cold atmospheric plasma (CAP) treatment is receiving more attention as potential sterilization method at relatively mild conditions but the exact mechanism of inactivation is still not fully understood. In this study, the biocidal effect by nitrogen CAP was determined for chemical (hypochlorite and hydrogen peroxide), physical (UV) and heat-resistant spores. The three different sporeformers used are Bacillus cereus a food-borne pathogen, and Bacillus atrophaeus and Geobacillus stearothermophilus that are used as biological indicators for validation of chemical sterilization and thermal processes, respectively. The different spores showed variation in their degree of inactivation by applied heat, hypochlorite, hydrogen peroxide, and UV treatments, whereas similar inactivation results were obtained with the different spores treated with nitrogen CAP. G. stearothermophilus spores displayed high resistance to heat, hypochlorite, hydrogen peroxide, while for UV treatment B. atrophaeus spores are most tolerant. Scanning electron microscopy analysis revealed distinct morphological changes for nitrogen CAP-treated B. cereus spores including etching effects and the appearance of rough spore surfaces, whereas morphology of spores treated with heat or disinfectants showed no such changes. Moreover, microscopy analysis revealed CAP-exposed B. cereus spores to turn phase grey conceivably because of water influx indicating damage of the spores, a phenomenon that was not observed for non-treated spores. In addition, data are supplied that exclude UV radiation as determinant of antimicrobial activity of nitrogen CAP. Overall, this study shows that nitrogen CAP treatment has a biocidal effect on selected Bacillus and Geobacillus spores associated with alterations in spore surface morphology and loss of spore integrity. Copyright © 2014 Elsevier Ltd. All rights reserved.

RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor.

PubMed

Mikulík, K; Janda, I; Weiser, J; Stastná, J; Jiránová, A

1984-12-03

Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time-ordered events. RNA and protein synthesis started during the first 5 min and net DNA synthesis at 60-70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4-5-S RNA were synthesized. Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C-labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.

Metabolites of Trichoderma species isolated from damp building materials.

PubMed

McMullin, David R; Renaud, Justin B; Barasubiye, Tharcisse; Sumarah, Mark W; Miller, J David

2017-07-01

Buildings that have been flooded often have high concentrations of Trichoderma spores in the air while drying. Inhaled spores and spore and mycelial fragments contain large amounts of fungal glucan and natural products that contribute to the symptoms associated with indoor mould exposures. In this study, we considered both small molecules and peptaibol profiles of T. atroviride, T. koningiopsis, T. citrinoviride, and T. harzianum strains obtained from damp buildings in eastern Canada. Twenty-residue peptaibols and sorbicillin-derived metabolites (1-6) including a new structure, (R)-vertinolide (1), were characterized from T. citrinoviride. Trichoderma koningiopsis produced several koninginins (7-10), trikoningin KA V, and the 11-residue lipopeptaibols trikoningin KB I and trikoningin KB II. Trichoderma atroviride biosynthesized a mixture of 19-residue trichorzianine-like peptaibols, whereas T. harzianum produced 18-residue trichokindin-like peptaibols and the 11-residue harzianin HB I that was subsequently identified from the studied T. citrinoviride strain. Two α-pyrones, 6-pentyl-pyran-2-one (11) and an oxidized analog (12), were produced by both T. atroviride and T. harzianum. Aside from exposure to low molecular weight natural products, inhalation of Trichoderma spores and mycelial fragments may result in exposure to membrane-disrupting peptaibols. This investigation contributes to a more comprehensive understanding of the biologically active natural products produced by fungi commonly found in damp buildings.

Distinction of broken cellular wall Ganoderma lucidum spores and G. lucidum spores using FTIR microspectroscopy

NASA Astrophysics Data System (ADS)

Chen, Xianliang; Liu, Xingcun; Sheng, Daping; Huang, Dake; Li, Weizu; Wang, Xin

2012-11-01

In this paper, FTIR microspectroscopy was used to identify broken cellular wall Ganoderma lucidum spores and G. lucidum spores. For IR spectra, broken cellular wall G. lucidum spores and G. lucidum spores were mainly different in the regions of 3000-2800, 1660-1600, 1400-1200 and 1100-1000 cm-1. For curve fitting, the results showed the differences in the protein secondary structures and the polysaccharide structures/content between broken cellular wall G. lucidum spores and G. lucidum spores. Moreover, the value of A1078/A1741 might be a potentially useful factor to distinguish broken cellular wall G. lucidum spores from G. lucidum spores. Additionally, FTIR microspectroscopy could identify broken cellular wall G. lucidum spores and G. lucidum spores accurately when it was combined with hierarchical cluster analysis. The result suggests FTIR microspectroscopy is very simple and efficient for distinction of broken cellular wall G. lucidum spores and G. lucidum spores. The result also indicates FTIR microspectroscopy may be useful for TCM identification.

Structure and function of CrACA1, the major PM-type Ca2+-ATPase, expressed at the peak of the gravity-directed trans-cell calcium current in spores of the fern Ceratopteris richardii.

PubMed

Bushart, T J; Cannon, A; Clark, G; Roux, S J

2014-01-01

Spores of the fern Ceratopteris richardii have proven to be a valuable single-cell system for studying gravity responses. The earliest cellular change directed by gravity in these cells is a trans-cell calcium current, which peaks near 10 h after the spores are induced to germinate. This current is needed for gravity-directed axis alignment, and its peak is coincident with the time period when gravity polarises the direction of subsequent nuclear migration and rhizoid growth. Transcriptomic analysis of genes expressed at the 10-h time point revealed several that encode proteins likely to be key components that either drive the current or regulate it. Notable among these is a plasma membrane (PM)-type Ca(2+) ATPase, CrACA1, whose activity pumping Ca(2+) out of cells is regulated by gravity. This report provides an initial characterisation of the structure and expression of this protein, and demonstrates its heterologous function complementing the K616 mutant of yeast, which is deficient in PM-type Ca(2+) pump activity. Gravity-induced changes in the trans-cell Ca(2+) current occur within seconds, a result consistent with the hypothesis that the force of gravity can rapidly alter the post-translational state of the channels and pumps that drive this current across spore cells. This report identifies a transporter likely to be a key driver of the current, CrACA1, and characterises the role of this protein in early germination and gravity-driven polarity fixation through analysis of expression levels, functional complementation and pharmacological treatments. These data, along with newly available transcriptomic data obtained at the 10-h time point, indicate that CrACA1 is present, functional and likely a major contributing component of the trans-cell Ca(2+) efflux. CrACA1 is not necessary for polar axis alignment, but pharmacological perturbations of it disrupt rhizoid development. These data support and help refine the post-translational modification model for gravity responses. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

Gene activity during germination of spores of the fern, Onoclea sensibilis. Cell-free translation analysis of mRNA of spores and the effect of alpha-amanitin on spore germination

NASA Technical Reports Server (NTRS)

Raghavan, V.

1992-01-01

Poly(A)-RNA fractions of dormant, dark-imbibed (non-germinating) and photoinduced (germinating) spores of Onoclea sensibilis were poor templates in the rabbit reticulocyte lysate protein synthesizing system, but the translational efficiency of poly(A)+RNA was considerably higher than that of unfractionated RNA. Poly(A)+RNA isolated from photoinduced spores had a consistently higher translational efficiency than poly(A)+RNA from dark-imbibed spores. Analysis of the translation products by one-dimensional polyacrylamide gel electrophoresis showed no qualitative differences in the mRNA populations of dormant, dark-imbibed, and photoinduced spores. However, poly(A)+RNA from dark-imbibed spores appeared to encode in vitro fewer detectable polypeptides at a reduced intensity than photoinduced spores. A DNA clone encoding the large subunit of maize ribulose bisphosphate carboxylase hybridized at strong to moderate intensity to RNA isolated from dark-imbibed spores, indicating the absence of mRNA degradation. Although alpha-amanitin did not inhibit the germination of spores, the drug prevented the elongation of the rhizoid and protonemal initial with a concomitant effect on the synthesis of poly(A)+RNA. These results are consistent with the view that some form of translational control involving stored mRNA operates during dark-imbibition and photoinduced germination of spores.

Analysis of the Effects of a gerP Mutation on the Germination of Spores of Bacillus subtilis

DTIC Science & Technology

2012-11-01

REPORT Analysis of the effects of a gerP mutation on the germination of spores of Bacillus subtilis 14. ABSTRACT 16. SECURITY CLASSIFICATION OF... Bacillus subtilis spores with a gerP mutation triggered spore germination via nutrient germinant receptors (GRs) slowly, although this defect was...gerP, Bacillus subtilis , dipicolinic acid Xuan Y. Butzin, Anthony J. Troiano, William H. Coleman, Keren K. Griffiths, Christopher J. Doona, Florence E

Nanoscale Structural and Mechanical Analysis of Bacillus anthracis Spores Inactivated with Rapid Dry Heating

PubMed Central

Felker, Daniel L.; Burggraf, Larry W.

2014-01-01

Effective killing of Bacillus anthracis spores is of paramount importance to antibioterrorism, food safety, environmental protection, and the medical device industry. Thus, a deeper understanding of the mechanisms of spore resistance and inactivation is highly desired for developing new strategies or improving the known methods for spore destruction. Previous studies have shown that spore inactivation mechanisms differ considerably depending upon the killing agents, such as heat (wet heat, dry heat), UV, ionizing radiation, and chemicals. It is believed that wet heat kills spores by inactivating critical enzymes, while dry heat kills spores by damaging their DNA. Many studies have focused on the biochemical aspects of spore inactivation by dry heat; few have investigated structural damages and changes in spore mechanical properties. In this study, we have inactivated Bacillus anthracis spores with rapid dry heating and performed nanoscale topographical and mechanical analysis of inactivated spores using atomic force microscopy (AFM). Our results revealed significant changes in spore morphology and nanomechanical properties after heat inactivation. In addition, we also found that these changes were different under different heating conditions that produced similar inactivation probabilities (high temperature for short exposure time versus low temperature for long exposure time). We attributed the differences to the differential thermal and mechanical stresses in the spore. The buildup of internal thermal and mechanical stresses may become prominent only in ultrafast, high-temperature heat inactivation when the experimental timescale is too short for heat-generated vapor to efficiently escape from the spore. Our results thus provide direct, visual evidences of the importance of thermal stresses and heat and mass transfer to spore inactivation by very rapid dry heating. PMID:24375142

Atmospheric mold spore counts in relation to meteorological parameters

NASA Astrophysics Data System (ADS)

Katial, R. K.; Zhang, Yiming; Jones, Richard H.; Dyer, Philip D.

Fungal spore counts of Cladosporium, Alternaria, and Epicoccum were studied during 8 years in Denver, Colorado. Fungal spore counts were obtained daily during the pollinating season by a Rotorod sampler. Weather data were obtained from the National Climatic Data Center. Daily averages of temperature, relative humidity, daily precipitation, barometric pressure, and wind speed were studied. A time series analysis was performed on the data to mathematically model the spore counts in relation to weather parameters. Using SAS PROC ARIMA software, a regression analysis was performed, regressing the spore counts on the weather variables assuming an autoregressive moving average (ARMA) error structure. Cladosporium was found to be positively correlated (P<0.02) with average daily temperature, relative humidity, and negatively correlated with precipitation. Alternaria and Epicoccum did not show increased predictability with weather variables. A mathematical model was derived for Cladosporium spore counts using the annual seasonal cycle and significant weather variables. The model for Alternaria and Epicoccum incorporated the annual seasonal cycle. Fungal spore counts can be modeled by time series analysis and related to meteorological parameters controlling for seasonallity; this modeling can provide estimates of exposure to fungal aeroallergens.

Distinction of broken cellular wall Ganoderma lucidum spores and G. lucidum spores using FTIR microspectroscopy.

PubMed

Chen, Xianliang; Liu, Xingcun; Sheng, Daping; Huang, Dake; Li, Weizu; Wang, Xin

2012-11-01

In this paper, FTIR microspectroscopy was used to identify broken cellular wall Ganoderma lucidum spores and G. lucidum spores. For IR spectra, broken cellular wall G. lucidum spores and G. lucidum spores were mainly different in the regions of 3000-2800, 1660-1600, 1400-1200 and 1100-1000 cm(-1). For curve fitting, the results showed the differences in the protein secondary structures and the polysaccharide structures/content between broken cellular wall G. lucidum spores and G. lucidum spores. Moreover, the value of A1078/A1741 might be a potentially useful factor to distinguish broken cellular wall G. lucidum spores from G. lucidum spores. Additionally, FTIR microspectroscopy could identify broken cellular wall G. lucidum spores and G. lucidum spores accurately when it was combined with hierarchical cluster analysis. The result suggests FTIR microspectroscopy is very simple and efficient for distinction of broken cellular wall G. lucidum spores and G. lucidum spores. The result also indicates FTIR microspectroscopy may be useful for TCM identification. Copyright © 2012 Elsevier B.V. All rights reserved.

Characterization of the yrbA Gene of Bacillus subtilis, Involved in Resistance and Germination of Spores

PubMed Central

Takamatsu, Hiromu; Kodama, Takeko; Nakayama, Tatsuo; Watabe, Kazuhito

1999-01-01

Insertional inactivation of the yrbA gene of Bacillus subtilis reduced the resistance of the mutant spores to lysozyme. The yrbA mutant spores lost their optical density at the same rate as the wild-type spores upon incubation with l-alanine but became only phase gray and did not swell. The response of the mutant spores to a combination of asparagine, glucose, fructose, and KCl was also extremely poor; in this medium yrbA spores exhibited only a small loss in optical density and gave a mixture of phase-bright, -gray, and -dark spores. Northern blot analysis of yrbA transcripts in various sig mutants indicated that yrbA was transcribed by RNA polymerase with ςE beginning at 2 h after the start of sporulation. The yrbA promoter was localized by primer extension analysis, and the sequences of the −35 (TCATAAC) and −10 (CATATGT) regions were similar to the consensus sequences of genes recognized by ςE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins solubilized from intact yrbA mutant spores showed an alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some other minor changes. Electron microscopic examination of yrbA spores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the appearance of an inner coat layer-like structure around the outside of the coat. This abnormal coat structure was also observed on the outside of the developing forespores of the yrbA mutant. These results suggest that YrbA is involved in assembly of some coat proteins which have roles in both spore lysozyme resistance and germination. PMID:10438771

Relationships among North American and Japanese Laetiporus isolates inferred from molecular phylogenetics and single-spore incompatibility reactions

Treesearch

Mark T. Banik; Daniel L. Lindner; Yuko Ota; Tsutomu Hattori

2010-01-01

Relationships were investigated among North American and Japanese isolates of Laetiporus using phylogenetic analysis of ITS sequences and single-spore isolate incompatibility. Single-spore isolate pairings revealed no significant compatibility between North American and Japanese isolates. ITS analysis revealed 12 clades within the core ...

Image Cytometric Analysis of Algal Spores for Evaluation of Antifouling Activities of Biocidal Agents.

PubMed

Il Koo, Bon; Lee, Yun-Soo; Seo, Mintae; Seok Choi, Hyung; Leng Seah, Geok; Nam, Taegu; Nam, Yoon Sung

2017-07-31

Chemical biocides have been widely used as marine antifouling agents, but their environmental toxicity impose regulatory restriction on their use. Although various surrogate antifouling biocides have been introduced, their comparative effectiveness has not been well investigated partly due to the difficulty of quantitative evaluation of their antifouling activity. Here we report an image cytometric method to quantitatively analyze the antifouling activities of seven commercial biocides using Ulva prolifera as a target organism, which is known to be a dominant marine species causing soft fouling. The number of spores settled on a substrate is determined through image analysis using the intrinsic fluorescence of chlorophylls in the spores. Pre-determined sets of size and shape of spores allow for the precise determination of the number of settled spores. The effects of biocide concentration and combination of different biocides on the spore settlement are examined. No significant morphological changes of Ulva spores are observed, but the amount of adhesive pad materials is appreciably decreased in the presence of biocides. It is revealed that the growth rate of Ulva is not directly correlated with the antifouling activities against the settlement of Ulva spores. This work suggests that image cytometric analysis is a very convenient, fast-processable method to directly analyze the antifouling effects of biocides and coating materials.

Intestinal disease of scattered mirror carp Cyprinus carpio caused by Thelohanellus kitauei and notes on the morphology and phylogeny of the myxosporean from Sichuan Province, southwest China

NASA Astrophysics Data System (ADS)

Ye, Lingtong; Lu, Mingmiao; Quan, Keyan; Li, Wenxiang; Zou, Hong; Wu, Shangong; Wang, Jiangyong; Wang, Guitang

2017-05-01

The mass mortality of pond-reared scattered mirror carp, Cyprinus carpio, caused by Thelohanellus kitauei, occurred at fish farms in Sichuan Province, southwest China. Morphological and molecular analyses were supplemented with histological evaluation of infected tissues to better understand the route of infection and the pathological effects of T. kitauei on the fish host. The intestine of the diseased host was full of large cysts of the myxosporean. The cysts range from 2 cm to 3.6 cm in diameter. Histopathology indicated that T. kitaue i first invaded the submucosa of the host intestine and then moved into the mucosa layers with the development of their spores, finally entering into the enteric cavity of the hosts after the disruption of mucosa layers. The pyriform spores of T. kitauei were surrounded by the transparent spore sheath, measuring 25.98 μm±0.95 μm in body length, 8.72 μm±0.51 μm in body width, and 7.86 μm±0.26 μm in body thickness. The single polar capsule was pyriform, measuring 14.73 μm±0.92 μm in length and 6.82 μm±0.45 μm in width, with eight to 10 turns of filament coils winding inside. Phylogenetic analysis based on the 18S small-subunit ribosomal DNA sequences indicated that minimal genetic differences were present between T. kitauei samples from South Korea and from China. Close affinity was found between the genus Thelohanellus and Myxobolus. Additionally, two polar capsule nuclei were found at the anterior end of the single polar capsule in spores of T. kitauei stained with hematoxylin and eosin, which suggested the separation of the genus Thelohanellus from Myxobolus.

High-throughput tetrad analysis.

PubMed

Ludlow, Catherine L; Scott, Adrian C; Cromie, Gareth A; Jeffery, Eric W; Sirr, Amy; May, Patrick; Lin, Jake; Gilbert, Teresa L; Hays, Michelle; Dudley, Aimée M

2013-07-01

Tetrad analysis has been a gold-standard genetic technique for several decades. Unfortunately, the need to manually isolate, disrupt and space tetrads has relegated its application to small-scale studies and limited its integration with high-throughput DNA sequencing technologies. We have developed a rapid, high-throughput method, called barcode-enabled sequencing of tetrads (BEST), that uses (i) a meiosis-specific GFP fusion protein to isolate tetrads by FACS and (ii) molecular barcodes that are read during genotyping to identify spores derived from the same tetrad. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. An individual researcher was able to isolate over 3,000 yeast tetrads in 3 h, an output equivalent to that of almost 1 month of manual dissection. BEST is transferable to other microorganisms for which meiotic mapping is significantly more laborious.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy.

PubMed

Fuchs, Felix M; Raguse, Marina; Fiebrandt, Marcel; Madela, Kazimierz; Awakowicz, Peter; Laue, Michael; Stapelmann, Katharina; Moeller, Ralf

2017-11-30

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

High temperature flow-through device for rapid solubilization and analysis

DOEpatents

West, Jason A. A. [Castro Valley, CA; Hukari, Kyle W [San Ramon, CA; Patel, Kamlesh D [Dublin, CA; Peterson, Kenneth A [Albuquerque, NM; Renzi, Ronald F [Tracy, CA

2009-09-22

Devices and methods for thermally lysing of biological material, for example vegetative bacterial cells and bacterial spores, are provided. Hot solution methods for solubilizing bacterial spores are described. Systems for direct analysis are disclosed including thermal lysers coupled to sample preparation stations. Integrated systems capable of performing sample lysis, labeling and protein fingerprint analysis of biological material, for example, vegetative bacterial cells, bacterial spores and viruses are provided.

High temperature flow-through device for rapid solubilization and analysis

DOEpatents

West, Jason A. A.; Hukari, Kyle W.; Patel, Kamlesh D.; Peterson, Kenneth A.; Renzi, Ronald F.

2013-04-23

Devices and methods for thermally lysing of biological material, for example vegetative bacterial cells and bacterial spores, are provided. Hot solution methods for solubilizing bacterial spores are described. Systems for direct analysis are disclosed including thermal lysers coupled to sample preparation stations. Integrated systems capable of performing sample lysis, labeling and protein fingerprint analysis of biological material, for example, vegetative bacterial cells, bacterial spores and viruses are provided.

Surface tension propulsion of fungal spores by use of microdroplets

NASA Astrophysics Data System (ADS)

Noblin, Xavier; Yang, Sylvia; Dumais, Jacques

2010-11-01

Most basidiomycete fungi (such as edible mushrooms) actively eject their spores. The process begins with the condensation of a water droplet at the base of the spore. The fusion of the droplet onto the spore creates a momentum that propels the spore forward. The use of surface tension for spore ejection offers a new paradigm to perform work at small length scales. However, this mechanism of force generation remains poorly understood. To elucidate how fungal spores make effective use of surface tension, we performed high-speed video imaging of spore ejection in Auricularia auricula and Sporobolomyces yeast, along with a detailed mechanical analysis of the spore ejection. We developed an explicit relation for the conversion of surface energy into kinetic energy during the coalescence process. The relation was validated with a simple artificial system.

Rapid identification of Bacillus anthracis spores in suspicious powder samples by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

PubMed

Dybwad, Marius; van der Laaken, Anton L; Blatny, Janet Martha; Paauw, Armand

2013-09-01

Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method's limit of B. anthracis detection was determined to be 2.5 × 10(6) spores, equivalent to a 55-μg sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min.

Rapid Identification of Bacillus anthracis Spores in Suspicious Powder Samples by Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS)

PubMed Central

van der Laaken, Anton L.; Blatny, Janet Martha; Paauw, Armand

2013-01-01

Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method's limit of B. anthracis detection was determined to be 2.5 × 106 spores, equivalent to a 55-μg sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min. PMID:23811517

Spatial and temporal distribution of Alternaria spores in the Iberian Peninsula atmosphere, and meteorological relationships: 1993-2009

NASA Astrophysics Data System (ADS)

Aira, María-Jesús; Rodríguez-Rajo, Francisco-Javier; Fernández-González, María; Seijo, Carmen; Elvira-Rendueles, Belén; Abreu, Ilda; Gutiérrez-Bustillo, Montserrat; Pérez-Sánchez, Elena; Oliveira, Manuela; Recio, Marta; Tormo, Rafael; Morales, Julia

2013-03-01

This paper provides an updated of airborne Alternaria spore spatial and temporal distribution patterns in the Iberian Peninsula, using a common non-viable volumetric sampling method. The highest mean annual spore counts were recorded in Sevilla (39,418 spores), Mérida (33,744) and Málaga (12,947), while other sampling stations never exceeded 5,000. The same cities also recorded the highest mean daily spore counts (Sevilla 109 spores m-3; Mérida 53 spores m-3 and Málaga 35 spores m-3) and the highest number of days on which counts exceeded the threshold levels required to trigger allergy symptoms (Sevilla 38 % and Mérida 30 % of days). Analysis of annual spore distribution patterns revealed either one or two peaks, depending on the location and prevailing climate of sampling stations. For all stations, average temperature was the weather parameter displaying the strongest positive correlation with airborne spore counts, whilst negative correlations were found for rainfall and relative humidity.

Spatial and temporal distribution of Alternaria spores in the Iberian Peninsula atmosphere, and meteorological relationships: 1993-2009.

PubMed

Aira, María-Jesús; Rodríguez-Rajo, Francisco-Javier; Fernández-González, María; Seijo, Carmen; Elvira-Rendueles, Belén; Abreu, Ilda; Gutiérrez-Bustillo, Montserrat; Pérez-Sánchez, Elena; Oliveira, Manuela; Recio, Marta; Tormo, Rafael; Morales, Julia

2013-03-01

This paper provides an updated of airborne Alternaria spore spatial and temporal distribution patterns in the Iberian Peninsula, using a common non-viable volumetric sampling method. The highest mean annual spore counts were recorded in Sevilla (39,418 spores), Mérida (33,744) and Málaga (12,947), while other sampling stations never exceeded 5,000. The same cities also recorded the highest mean daily spore counts (Sevilla 109 spores m(-3); Mérida 53 spores m(-3) and Málaga 35 spores m(-3)) and the highest number of days on which counts exceeded the threshold levels required to trigger allergy symptoms (Sevilla 38 % and Mérida 30 % of days). Analysis of annual spore distribution patterns revealed either one or two peaks, depending on the location and prevailing climate of sampling stations. For all stations, average temperature was the weather parameter displaying the strongest positive correlation with airborne spore counts, whilst negative correlations were found for rainfall and relative humidity.

The characterisation of Bacillus spores occurring in the manufacturing of (low acid) canned products.

PubMed

Oomes, S J C M; van Zuijlen, A C M; Hehenkamp, J O; Witsenboer, H; van der Vossen, J M B M; Brul, S

2007-11-30

Spore-forming bacteria can be a problem in the food industry, especially in the canning industry. Spores present in ingredients or present in the processing environment severely challenge the preservation process since their thermal resistance may be very high. We therefore asked the question which bacterial spore formers are found in a typical soup manufacturing plant, where they originate from and what the thermal resistance of their spores is. To answer these questions molecular techniques for bacterial species and strain identification were used as well as a protocol for the assessment of spore heat stress resistance based on the Kooiman method. The data indicate the existence and physiological cause of the high thermal resistance of spores of many of the occurring species. In particular it shows that ingredients used in soup manufacturing are a rich source of high thermal resistant spores and that sporulation in the presence of ingredients rich in divalent metal ions exerts a strong influence on spore heat resistance. It was also indicated that Bacillus spores may well be able to germinate and resporulate during manufacturing i.e. through growth and sporulation in line. Both these spores and those originating from the ingredients were able to survive certain thermal processing settings. Species identity was confirmed using fatty acid analysis, 16SrRNA gene sequencing and DNA-DNA hybridisation. Finally, molecular typing experiments using Ribotyping and AFLP analysis show that strains within the various Bacillus species can be clustered according to the thermal resistance properties of their spores. AFLP performed slightly better than Ribotyping. The data proofed to be useful for the generation of strain specific probes. Protocols to validate these probes in routine identification and innovation aimed at tailor made heat processing in soup manufacturing have been formulated.

Effects of meteorological conditions on spore plumes

NASA Astrophysics Data System (ADS)

Burch, M.; Levetin, E.

2002-05-01

Fungal spores are an ever-present component of the atmosphere, and have long been known to trigger asthma and hay fever symptoms in sensitive individuals. The atmosphere around Tulsa has been monitored for airborne spores and pollen with Burkard spore traps at several sampling stations. This study involved the examination of the hourly spore concentrations on days that had average daily concentrations near 50,000 spores/m3 or greater. Hourly concentrations of Cladosporium, Alternaria, Epicoccum, Curvularia, Pithomyces, Drechslera, smut spores, ascospores, basidiospores, other, and total spores were determined on 4 days at three sites and then correlated with hourly meteorological data including temperature, rainfall, wind speed, dew point, air pressure, and wind direction. On each of these days there was a spore plume, a phenomenon in which spore concentrations increased dramatically over a very short period of time. Spore plumes generally occurred near midday, and concentrations were seen to increase from lows around 20,000 total spores/m3 to highs over 170,000 total spores/m3 in 2 h. Multiple regression analysis of the data indicated that increases in temperature, dew point, and air pressure correlated with the increase in spore concentrations, but no single weather variable predicted the appearance of a spore plume. The proper combination of changes in these meteorological parameters that result in a spore plume may be due to the changing weather conditions associated with thunderstorms, as on 3 of the 4 days when spore plumes occurred there were thunderstorms later that evening. The occurrence of spore plumes may have clinical significance, because other studies have shown that sensitization to certain spore types can occur during exposure to high spore concentrations.

Bonamia perspora n. sp. (Haplosporidia), a parasite of the oyster Ostreola equestris, is the first Bonamia species known to produce spores.

PubMed

Carnegie, Ryan B; Burreson, Eugene M; Hine, P Mike; Stokes, Nancy A; Audemard, Corinne; Bishop, Melanie J; Peterson, Charles H

2006-01-01

Examination of the oyster Ostreola equestris as a potential reservoir host for a species of Bonamia discovered in Crassostrea ariakensis in North Carolina (NC), USA, revealed a second novel Bonamia sp. Histopathology, electron microscopy, and molecular phylogenetic analysis support the designation of a new parasite species, Bonamia perspora n. sp., which is the first Bonamia species shown to produce a typical haplosporidian spore with an orifice and hinged operculum. Spores were confirmed to be from B. perspora by fluorescent in situ hybridization. Bonamia perspora was found at Morehead City and Wilmington, NC, with an overall prevalence of 1.4% (31/2,144). Uninucleate, plasmodial, and sporogonic stages occurred almost exclusively in connective tissues; uninucleate stages (2-6 microm) were rarely observed in hemocytes. Spores were 4.3-6.4 microm in length. Ultrastructurally, uninucleate, diplokaryotic, and plasmodial stages resembled those of other spore-forming haplosporidians, but few haplosporosomes were present, and plasmodia were small. Spore ornamentation consisted of spore wall-derived, thin, flat ribbons that emerged haphazardly around the spore, and which terminated in what appeared to be four-pronged caps. Number of ribbons per spore ranged from 15 to 30, and their length ranged from 1.0 to 3.4 microm. Parsimony analysis identified B. perspora as a sister species to Bonamia ostreae.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Ziyu; Aryal, Uma K.; Shukla, Anil

ALG3 is a Family 58 glycosyltransferase enzyme involved in early N-linked glycan synthesis. Here, we investigated the effect of the alg3 gene disruption on growth, development, metabolism, and protein secretion in Aspergillus niger. The alg3 gene deletion resulted in a significant reduction of growth on complete (CM) and potato dextrose agar (PDA) media and a substantial reduction of spore production on CM. It also delayed spore germination in the liquid cultures of both CM and PDA media, but led to a significant accumulation of red pigment on both CM and liquid modified minimal medium (MM) supplemented with yeast extract. Themore » relative abundance of 55 proteins of the total 190 proteins identified in the secretome was significantly different as a result of alg3 gene deletion. Comparison of a Trichoderma reesei cellobiohydrolase (Cel7A) heterologously expressed in A. niger parental and Δalg3 strains showed that the recombinant Cel7A expressed in the mutant background was smaller in size than that from the parental strains. This study suggests that ALG3 is critical for growth and development, pigment production, and protein secretion in A. niger. Functional analysis of recombinant Cel7A with aberrant glycosylation demonstrates the feasibility of this alternative approach to evaluate the role of N-linked glycosylation in glycoprotein secretion and function.« less

Ricin-B-lectin enhances microsporidia Nosema bombycis infection in BmN cells from silkworm Bombyx mori.

PubMed

Liu, Han; Li, Mingqian; Cai, Shunfeng; He, Xinyi; Shao, Yongqi; Lu, Xingmeng

2016-11-01

Nosema bombycis is an obligate intracellular parasitic fungus that utilizes a distinctive mechanism to infect Bombyx mori Spore germination can be used for host cell invasion; however, the detailed mechanism remains to be elucidated. The ricin-B-lectin (RBL) gene is significantly differentially regulated after N. bombycis spore germination, and NbRBL might play roles in spore germination and infection. In this study, the biological function of NbRBL was examined. Protein sequence analysis showed that NbRBL is a secreted protein that attaches to carbohydrates. The relative expression level of the NbRBL gene was low during the first 30 h post-infection (hpi) in BmN cells, and high expression was detected from 42 hpi. Gene cloning, prokaryotic expression, and antibody preparation for NbRBL were performed. NbRBL was detected in total and secreted proteins using western blot analysis. Subcellular localization analysis showed that NbRBL is an intracellular protein. Spore adherence and infection assays showed that NbRBL could enhance spore adhesion to BmN cells; the proliferative activities of BmN cells incubated with anti-NbRBL were higher than those in negative control groups after N. bombycis infection; and the treatment groups showed less damage from spore invasion. We therefore, propose that NbRBL is released during spore germination, enhances spore adhesion to BmN cells, and contributes to spore invasion. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Purification and Characterization of Plantaricin JLA-9: A Novel Bacteriocin against Bacillus spp. Produced by Lactobacillus plantarum JLA-9 from Suan-Tsai, a Traditional Chinese Fermented Cabbage.

PubMed

Zhao, Shengming; Han, Jinzhi; Bie, Xiaomei; Lu, Zhaoxin; Zhang, Chong; Lv, Fengxia

2016-04-06

Bacteriocins are ribosomally synthesized peptides with antimicrobial activity produced by numerous bacteria. A novel bacteriocin-producing strain, Lactobacillus plantarum JLA-9, isolated from Suan-Tsai, a traditional Chinese fermented cabbage, was screened and identified by its physiobiochemical characteristics and 16S rDNA sequence analysis. A new bacteriocin, designated plantaricin JLA-9, was purified using butanol extraction, gel filtration, and reverse-phase high-performance liquid chromatography. The molecular mass of plantaricin JLA-9 was shown to be 1044 Da by MALDI-TOF-MS analyses. The amino acid sequence of plantaricin JLA-9 was predicted to be FWQKMSFA by MALDI-TOF-MS/MS, which was confirmed by Edman degradation. This bacteriocin exhibited broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, especially Bacillus spp., high thermal stability (20 min, 121 °C), and narrow pH stability (pH 2.0-7.0). It was sensitive to α-chymotrypsin, pepsin, alkaline protease, and papain. The mode of action of this bacteriocin responsible for outgrowth inhibition of Bacillus cereus spores was studied. Plantaricin JLA-9 had no detectable effects on germination initiation over 1 h on monitoring the hydration, heat resistance, and 2,6-pyridinedicarboxylic acid (DPA) release of spores. Rather, germination initiation is a prerequisite for the action of plantaricin JLA-9. Plantaricin JLA-9 inhibited growth by preventing the establishment of oxidative metabolism and disrupting membrane integrity in germinating spores within 2 h. The results suggest that plantaricin JLA-9 has potential applications in the control of Bacillus spp. in the food industry.

Weed control and cover crop management affect mycorrhizal colonization of grapevine roots and arbuscular mycorrhizal fungal spore populations in a California vineyard.

PubMed

Baumgartner, Kendra; Smith, Richard F; Bettiga, Larry

2005-03-01

Arbuscular mycorrhizal (AM) fungi naturally colonize grapevines in California vineyards. Weed control and cover cropping may affect AM fungi directly, through destruction of extraradical hyphae by soil disruption, or indirectly, through effects on populations of mycorrhizal weeds and cover crops. We examined the effects of weed control (cultivation, post-emergence herbicides, pre-emergence herbicides) and cover crops (Secale cereale cv. Merced rye, x Triticosecale cv.Trios 102) on AM fungi in a Central Coast vineyard. Seasonal changes in grapevine mycorrhizal colonization differed among weed control treatments, but did not correspond with seasonal changes in total weed frequency. Differences in grapevine colonization among weed control treatments may be due to differences in mycorrhizal status and/or AM fungal species composition among dominant weed species. Cover crops had no effect on grapevine mycorrhizal colonization, despite higher spring spore populations in cover cropped middles compared to bare middles. Cover crops were mycorrhizal and shared four AM fungal species (Glomus aggregatum, G. etunicatum, G. mosseae, G. scintillans) in common with grapevines. Lack of contact between grapevine roots and cover crop roots may have prevented grapevines from accessing higher spore populations in the middles.

Annual distribution of allergenic fungal spores in atmospheric particulate matter in the Eastern Mediterranean; a comparative study between ergosterol and quantitative PCR analysis

NASA Astrophysics Data System (ADS)

Lang-Yona, N.; Dannemiller, K.; Yamamoto, N.; Burshtein, N.; Peccia, J.; Yarden, O.; Rudich, Y.

2012-03-01

Airborne fungal spores are an important fraction of atmospheric particulate matter and are major causative agents of allergenic and infectious diseases. Predicting the variability and species of allergy-causing fungal spores requires detailed and reliable methods for identification and quantification. There are diverse methods for their detection in the atmosphere and in the indoor environments; yet, it is important to optimize suitable methods for characterization of fungal spores in atmospheric samples. In this study we sampled and characterized total and specific airborne fungal spores from PM10 samples collected in Rehovot, Israel over an entire year. The total fungal spore concentrations vary throughout the year although the species variability was nearly the same. Seasonal equivalent spore concentrations analyzed by real-time quantitative-PCR-based methods were fall > winter > spring > summer. Reported concentrations based on ergosterol analysis for the same samples were and fall > spring > winter > summer. Correlation between the two analytical methods was found only for the spring season. These poor associations may be due to the per-spore ergosterol variations that arise from both varying production rates, as well as molecular degradation of ergosterol. While conversion of genome copies to spore concentration is not yet straightforward, the potential for improving this conversion and the ability of qPCR to identify groups of fungi or specific species makes this method preferable for environmental spore quantification. Identifying tools for establishing the relation between the presence of species and the actual ability to induce allergies is still needed in order to predict the effect on human health.

Annual distribution of allergenic fungal spores in atmospheric particulate matter in the eastern mediterranean; a comparative study between ergosterol and quantitative PCR analysis

NASA Astrophysics Data System (ADS)

Lang-Yona, N.; Dannemiller, K.; Yamamoto, N.; Burshtein, N.; Peccia, J.; Yarden, O.; Rudich, Y.

2011-10-01

Airborne fungal spores are an important fraction of atmospheric particulate matter and are major causative agents of allergenic and infectious diseases. Predicting the variability and species of allergy-causing fungal spores requires detailed and reliable methods for identification and quantification. There are diverse methods for their detection in the atmosphere and in the indoor environments; yet, it is important to optimize suitable methods for characterization of fungal spores in atmospheric samples. In this study we sampled and characterized total and specific airborne fungal spores from PM10 samples collected in Rohovot, Israel over an entire year. The total fungal spore concentrations vary throughout the year although the species variability was nearly the same. Seasonal equivalent spore concentrations analyzed by real-time quantitative-PCR-based methods were fall > winter > spring > summer. Reported concentrations based on ergosterol analysis for the same samples were and fall > spring > winter > summer. Correlation between the two analytical methods was found only for the spring season. These poor associations may be due to the per-spore ergosterol variations that arise from both varying production rates, as well as molecular degradation of ergosterol. While conversion of genome copies to spore concentration is not yet straightforward, the potential for improving this conversion and the ability of qPCR to identify groups of fungi or specific species makes this method preferable for environmental spore quantification. Identifying tools for establishing the relation between the presence of species and the actual ability to induce allergies is still needed in order to predict the effect on human health.

Decontamination of materials contaminated with Bacillus anthracis and Bacillus thuringiensis Al Hakam spores using PES-Solid, a solid source of peracetic acid.

PubMed

Buhr, T L; Wells, C M; Young, A A; Minter, Z A; Johnson, C A; Payne, A N; McPherson, D C

2013-08-01

To develop test methods and evaluate survival of Bacillus anthracis Ames, B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores after exposure to PES-Solid (a solid source of peracetic acid), including PES-Solid formulations with bacteriostatic surfactants. Spores (≥ 7 logs) were dried on seven different test materials and treated with three different PES-Solid formulations (or preneutralized controls) at room temperature for 15 min. There was either no spore survival or less than 1 log (<10 spores) of spore survival in 56 of 63 test combinations (strain, formulation and substrate). Less than 2.7 logs (<180 spores) survived in the remaining seven test combinations. The highest spore survival rates were seen on water-dispersible chemical agent resistant coating (CARC-W) and Naval ship topcoat (NTC). Electron microscopy and Coulter analysis showed that all spore structures were intact after spore inactivation with PES-Solid. Three PES-Solid formulations inactivated Bacillus spores that were dried on seven different materials. A test method was developed to show that PES-Solid formulations effectively inactivate Bacillus spores on different materials. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

A Spore Coat Protein, CotS, of Bacillus subtilis Is Synthesized under the Regulation of ςK and GerE during Development and Is Located in the Inner Coat Layer of Spores

PubMed Central

Takamatsu, Hiromu; Chikahiro, Yukari; Kodama, Takeko; Koide, Hidekatsu; Kozuka, Satoshi; Tochikubo, Kunio; Watabe, Kazuhito

1998-01-01

The spore coat of Bacillus subtilis has a unique morphology and consists of polypeptides of different sizes, whose synthesis and assembly are precisely regulated by a cascade of transcription factors and regulatory proteins. We examined the factors that regulate cotS gene expression and CotS assembly into the coat layer of B. subtilis by Northern blot and Western blot analysis. Transcription of cotS mRNA was not detected in sporulating cells of ςK and gerE mutants by Northern blot analysis. By Western blot analysis using anti-CotS antibody, CotS was first detected in protein samples solubilized from wild-type cells at 5 h after the start of sporulation. CotS was not detected in the vegetative cells and spores of a gerE mutant or in the spores of mutants deficient in ςE, ςF, ςG, or ςK. CotS was detected in the sporangium but not in the spores of a cotE mutant. The sequence of the promoter region of cotS was similar to the consensus sequences for binding of ςK and GerE. These results demonstrate that ςK and GerE are required for cotS expression and that CotE is essential for the assembly of CotS in the coat. Immunoelectron microscopic observation using anti-CotS antibody revealed that CotS is located within the spore coat, in particular in the inner coats of dormant spores. PMID:9603889

Quantification of Nonproteolytic Clostridium botulinum Spore Loads in Food Materials.

PubMed

Barker, Gary C; Malakar, Pradeep K; Plowman, June; Peck, Michael W

2016-01-04

We have produced data and developed analysis to build representations for the concentration of spores of nonproteolytic Clostridium botulinum in materials that are used during the manufacture of minimally processed chilled foods in the United Kingdom. Food materials are categorized into homogenous groups which include meat, fish, shellfish, cereals, fresh plant material, dairy liquid, dairy nonliquid, mushroom and fungi, and dried herbs and spices. Models are constructed in a Bayesian framework and represent a combination of information from a literature survey of spore loads from positive-control experiments that establish a detection limit and from dedicated microbiological tests for real food materials. The detection of nonproteolytic C. botulinum employed an optimized protocol that combines selective enrichment culture with multiplex PCR, and the majority of tests on food materials were negative. Posterior beliefs about spore loads center on a concentration range of 1 to 10 spores kg(-1). Posterior beliefs for larger spore loads were most significant for dried herbs and spices and were most sensitive to the detailed results from control experiments. Probability distributions for spore loads are represented in a convenient form that can be used for numerical analysis and risk assessments. Copyright © 2016 Barker et al.

Quantification of Nonproteolytic Clostridium botulinum Spore Loads in Food Materials

PubMed Central

Barker, Gary C.; Malakar, Pradeep K.; Plowman, June

2016-01-01

We have produced data and developed analysis to build representations for the concentration of spores of nonproteolytic Clostridium botulinum in materials that are used during the manufacture of minimally processed chilled foods in the United Kingdom. Food materials are categorized into homogenous groups which include meat, fish, shellfish, cereals, fresh plant material, dairy liquid, dairy nonliquid, mushroom and fungi, and dried herbs and spices. Models are constructed in a Bayesian framework and represent a combination of information from a literature survey of spore loads from positive-control experiments that establish a detection limit and from dedicated microbiological tests for real food materials. The detection of nonproteolytic C. botulinum employed an optimized protocol that combines selective enrichment culture with multiplex PCR, and the majority of tests on food materials were negative. Posterior beliefs about spore loads center on a concentration range of 1 to 10 spores kg−1. Posterior beliefs for larger spore loads were most significant for dried herbs and spices and were most sensitive to the detailed results from control experiments. Probability distributions for spore loads are represented in a convenient form that can be used for numerical analysis and risk assessments. PMID:26729721

Systematic Analysis of γ-Aminobutyric Acid (GABA) Metabolism and Function in the Social Amoeba Dictyostelium discoideum*

PubMed Central

Wu, Yuantai; Janetopoulos, Chris

2013-01-01

While GABA has been suggested to regulate spore encapsulation in the social amoeba Dictyostelium discoideum, the metabolic profile and other potential functions of GABA during development remain unclear. In this study, we investigated the homeostasis of GABA metabolism by disrupting genes related to GABA metabolism and signaling. Extracellular levels of GABA are tightly regulated during early development, and GABA is generated by the glutamate decarboxylase, GadB, during growth and in early development. However, overexpression of the prespore-specific homologue, GadA, in the presence of GadB reduces production of extracellular GABA. Perturbation of extracellular GABA levels delays the process of aggregation. Cytosolic GABA is degraded by the GABA transaminase, GabT, in the mitochondria. Disruption of a putative vesicular GABA transporter (vGAT) homologue DdvGAT reduces secreted GABA. We identified the GABAB receptor-like family member GrlB as the major GABA receptor during early development, and either disruption or overexpression of GrlB delays aggregation. This delay is likely the result of an abolished pre-starvation response and late expression of several “early” developmental genes. Distinct genes are employed for GABA generation during sporulation. During sporulation, GadA alone is required for generating GABA and DdvGAT is likely responsible for GABA secretion. GrlE but not GrlB is the GABA receptor during late development. PMID:23548898

Composite Sampling Approaches for Bacillus anthracis Surrogate Extracted from Soil

PubMed Central

France, Brian; Bell, William; Chang, Emily; Scholten, Trudy

2015-01-01

Any release of anthrax spores in the U.S. would require action to decontaminate the site and restore its use and operations as rapidly as possible. The remediation activity would require environmental sampling, both initially to determine the extent of contamination (hazard mapping) and post-decon to determine that the site is free of contamination (clearance sampling). Whether the spore contamination is within a building or outdoors, collecting and analyzing what could be thousands of samples can become the factor that limits the pace of restoring operations. To address this sampling and analysis bottleneck and decrease the time needed to recover from an anthrax contamination event, this study investigates the use of composite sampling. Pooling or compositing of samples is an established technique to reduce the number of analyses required, and its use for anthrax spore sampling has recently been investigated. However, use of composite sampling in an anthrax spore remediation event will require well-documented and accepted methods. In particular, previous composite sampling studies have focused on sampling from hard surfaces; data on soil sampling are required to extend the procedure to outdoor use. Further, we must consider whether combining liquid samples, thus increasing the volume, lowers the sensitivity of detection and produces false negatives. In this study, methods to composite bacterial spore samples from soil are demonstrated. B. subtilis spore suspensions were used as a surrogate for anthrax spores. Two soils (Arizona Test Dust and sterilized potting soil) were contaminated and spore recovery with composites was shown to match individual sample performance. Results show that dilution can be overcome by concentrating bacterial spores using standard filtration methods. This study shows that composite sampling can be a viable method of pooling samples to reduce the number of analysis that must be performed during anthrax spore remediation. PMID:26714315

Fast analysis of triterpenoids in Ganoderma lucidum spores by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry.

PubMed

Yan, Zhou; Xia, Bing; Qiu, Ming Hua; Li Sheng, Ding; Xu, Hong Xi

2013-11-01

A rapid and reliable method was established for simultaneous determination of main triterpenoids in Ganoderma lucidum spores using ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-TQ-MS). The established method was validated in terms of linearity, sensitivity, precision, accuracy and stability, and was successfully applied to determine the contents of 10 main triterpenoids in different batches of G. lucidum spores. The analysis results showed that moderate levels of triterpenoids were found in G. lucidum spores. In addition, a MS full scan with a daughter ion scan experiment was performed to identify the potential derivatives of triterpenoids present in G. lucidum spores. As a result, a total of 22 triterpenoids from different G. lucidum spores were unequivocally or tentatively identified via comparisons with authentic standards and literatures. This method provides both qualitative and quantitative results without the need for repetitive UPLC-MS analyses, thereby increasing efficiency and productivity, making it suitable for high-throughput applications. Copyright © 2013 John Wiley & Sons, Ltd.

47 annual records of allergenic fungi spore: predictive models from the NW Iberian Peninsula.

PubMed

Aira, M Jesus; Rodriguez-Rajo, F; Jato, Victoria

2008-01-01

An analysis was carried out of the atmospheric representivity of Cladosporium and Alternaria spores in the north-western Iberian Peninsula, registering mean annual concentrations in excess of 300,000 spores/m(3). During the main sporulation period, the highest average daily concentrations corresponded to Cladosporium herbarum type (1,197 spores/m(3)) while the highest daily value was 7,556 spores/m(3) (Cladosporium cladosporioides type). Alternaria only represents between 0.1-1% of the total spores identified. In these spore types, the intraday variation was more acute inland than along the coastline due to oceanic influence. In the predictive models proposed that use the meteorological parameters with which a higher correlation was obtained (mean and maximum temperature) as predictive variables, it was seen that the predicted values did not reveal any significant differences as compared to those observed in 2006, data that was only used for verification purposes.

Most Probable Number Rapid Viability PCR Method to Detect Viable Spores of Bacillus anthracis in Swab Samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Letant, S E; Kane, S R; Murphy, G A

2008-05-30

This note presents a comparison of Most-Probable-Number Rapid Viability (MPN-RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs generated by the Centers for Disease Control and Prevention (CDC) for a multi-center validation study aimed at testing environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were in statistical agreement with the CDC conventional culture method for all three levels of spores tested (10{sup 4}, 10{sup 2}, and 10 spores) even in the presence ofmore » dirt. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols.« less

A study of Ganoderma lucidum spores by FTIR microspectroscopy

NASA Astrophysics Data System (ADS)

Wang, Xin; Chen, Xianliang; Qi, Zeming; Liu, Xingcun; Li, Weizu; Wang, Shengyi

2012-06-01

In order to obtain unique information of Ganoderma lucidum spores, FTIR microspectroscopy was used to study G. lucidum spores from Anhui Province (A), Liaoning Province (B) and Shangdong Province (C) of China. IR micro-spectra were acquired with high-resolution and well-reproducibility. The IR spectra of G. lucidum spores from different areas were similar and mainly made up of the absorption bands of polysaccharide, sterols, proteins, fatty acids, etc. The results of curve fitting indicated the protein secondary structures were dissimilar among the above G. lucidum spores. To identify G. lucidum spores from different areas, the H1078/H1640 value might be a potentially useful factor, furthermore FTIR microspectroscopy could realize this identification efficiently with the help of hierarchical cluster analysis. The result indicates FTIR microspectroscopy is an efficient tool for identification of G. lucidum spores from different areas. The result also suggests FTIR microspectroscopy is a potentially useful tool for the study of TCM.

Cytological and Proteomic Analyses of Osmunda cinnamomea Germinating Spores Reveal Characteristics of Fern Spore Germination and Rhizoid Tip Growth.

PubMed

Suo, Jinwei; Zhao, Qi; Zhang, Zhengxiu; Chen, Sixue; Cao, Jian'guo; Liu, Guanjun; Wei, Xing; Wang, Tai; Yang, Chuanping; Dai, Shaojun

2015-09-01

Fern spore is a good single-cell model for studying the sophisticated molecular networks in asymmetric cell division, differentiation, and polar growth. Osmunda cinnamomea L. var. asiatica is one of the oldest fern species with typical separate-growing trophophyll and sporophyll. The chlorophyllous spores generated from sporophyll can germinate without dormancy. In this study, the spore ultrastructure, antioxidant enzyme activities, as well as protein and gene expression patterns were analyzed in the course of spore germination at five typical stages (i.e. mature spores, rehydrated spores, double-celled spores, germinated spores, and spores with protonemal cells). Proteomic analysis revealed 113 differentially expressed proteins, which were mainly involved in photosynthesis, reserve mobilization, energy supplying, protein synthesis and turnover, reactive oxygen species scavenging, signaling, and cell structure modulation. The presence of multiple proteoforms of 25 differentially expressed proteins implies that post-translational modification may play important roles in spore germination. The dynamic patterns of proteins and their encoding genes exhibited specific characteristics in the processes of cell division and rhizoid tip growth, which include heterotrophic and autotrophic metabolisms, de novo protein synthesis and active protein turnover, reactive oxygen species and hormone (brassinosteroid and ethylene) signaling, and vesicle trafficking and cytoskeleton dynamic. In addition, the function skew of proteins in fern spores highlights the unique and common mechanisms when compared with evolutionarily divergent spermatophyte pollen. These findings provide an improved understanding of the typical single-celled asymmetric division and polar growth during fern spore germination. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of endo-1,3-1,4-β-glucanases in GH family 12 from Magnaporthe oryzae.

PubMed

Takeda, Takumi; Takahashi, Machiko; Nakanishi-Masuno, Tsugumi; Nakano, Yuki; Saitoh, Hiromasa; Hirabuchi, Akiko; Fujisawa, Shizuko; Terauchi, Ryohei

2010-11-01

We have cloned three putative endoglucanase cDNAs, designated MoCel12A, MoCel12B, and MoCel12C, from Magnaporthe oryzae. The deduced peptide sequences of both MoCel12A and MoCel12B contain secretion signal peptides and a catalytic core domain that classify them into GH subfamily 12-1. In contrast, the deduced peptide sequence of MoCel12C consists of a signal peptide, a catalytic core domain, and a fungal-type carbohydrate binding module belonging to GH subfamily 12-2. Although most GH family 12 endoglucanases hydrolyze β-1,4-glucans such as carboxymethylcellulose or phosphoric acid-swollen cellulose, MoCel12A that was prepared by overexpression in M. oryzae and Brevibacillus choshinensis hydrolyzed specifically 1,3-1,4-β-glucans, such as barley β-glucan and lichenan. The specific activity of MoCel12A overexpressed in M. oryzae was about 20 times higher than that prepared from B. choshinensis. Furthermore, MoCel12B prepared by overexpression in B. choshinensis also revealed preferential hydrolysis of endo-1,3-1,4-β-glucans with limited hydrolysis on carboxymethylcellulose. In comparison with MoCel12A, the activity of MoCel12B was more stable under alkaline conditions. Levels of mRNA encoding MoCel12A were constitutively high during infection and spore formation. The overexpression and disruption of the MoCel12A gene did not affect germination, appressorium formation, or invasion rate; however, M. oryzae overexpressing MoCel12A produced larger numbers of spores than the wild type or a mutant in which the MoCel12A gene was disrupted. These results suggest that MoCel12A functions in part to hydrolyze 1,3-1,4-β-glucan during infection and spore formation.

A lost link between a flightless parrot and a parasitic plant and the potential role of coprolites in conservation paleobiology.

PubMed

Wood, Jamie R; Wilmshurst, Janet M; Worthy, Trevor H; Holzapfel, Avi S; Cooper, Alan

2012-12-01

Late Quaternary extinctions and population fragmentations have severely disrupted animal-plant interactions globally. Detection of disrupted interactions often relies on anachronistic plant characteristics, such as spines in the absence of large herbivores or large fruit without dispersers. However, obvious anachronisms are relatively uncommon, and it can be difficult to prove a direct link between the anachronism and a particular faunal taxon. Analysis of coprolites (fossil feces) provides a novel way of exposing lost interactions between animals (depositors) and consumed organisms. We analyzed ancient DNA to show that a coprolite from the South Island of New Zealand was deposited by the rare and threatened kakapo (Strigops habroptilus), a large, nocturnal, flightless parrot. When we analyzed the pollen and spore content of the coprolite, we found pollen from the cryptic root-parasite Dactylanthus taylorii. The relatively high abundance (8.9% of total pollen and spores) of this zoophilous pollen type in the coprolite supports the hypothesis of a former direct feeding interaction between kakapo and D. taylorii. The ranges of both species have contracted substantially since human settlement, and their present distributions no longer overlap. Currently, the lesser short-tailed bat (Mystacina tuberculata) is the only known native pollinator of D. taylorii, but our finding raises the possibility that birds, and other small fauna, could have once fed on and pollinated the plant. If confirmed, through experimental work and observations, this finding may inform conservation of the plant. For example, it may be possible to translocate D. taylorii to predator-free offshore islands that lack bats but have thriving populations of endemic nectar-feeding birds. The study of coprolites of rare or extinct taxonomic groups provides a unique way forward to expand existing knowledge of lost plant and animal interactions and to identify pollination and dispersal syndromes. This approach of linking paleobiology with neoecology offers significant untapped potential to help inform conservation and restoration plans. ©2012 Society for Conservation Biology.

MreB of Streptomyces coelicolor is not essential for vegetative growth but is required for the integrity of aerial hyphae and spores.

PubMed

Mazza, Paola; Noens, Elke E; Schirner, Kathrin; Grantcharova, Nina; Mommaas, A Mieke; Koerten, Henk K; Muth, Günther; Flärdh, Klas; van Wezel, Gilles P; Wohlleben, Wolfgang

2006-05-01

MreB forms a cytoskeleton in many rod-shaped bacteria which is involved in cell shape determination and chromosome segregation. PCR-based and Southern analysis of various actinomycetes, supported by analysis of genome sequences, revealed mreB homologues only in genera that form an aerial mycelium and sporulate. We analysed MreB in one such organism, Streptomyces coelicolor. Ectopic overexpression of mreB impaired growth, and caused swellings and lysis of hyphae. A null mutant with apparently normal vegetative growth was generated. However, aerial hyphae of this mutant were swelling and lysing; spores doubled their volume and lost their characteristic resistance to stress conditions. Loss of cell wall consistency was observed in MreB-depleted spores by transmission electron microscopy. An MreB-EGFP fusion was constructed to localize MreB in the mycelium. No clearly localized signal was seen in vegetative mycelium. However, strong fluorescence was observed at the septa of sporulating aerial hyphae, then as bipolar foci in young spores, and finally in a ring- or shell-like pattern inside the spores. Immunogold electron microscopy using MreB-specific antibodies revealed that MreB is located immediately underneath the internal spore wall. Thus, MreB is not essential for vegetative growth of S. coelicolor, but exerts its function in the formation of environmentally stable spores, and appears to primarily influence the assembly of the spore cell wall.

Genetic Diversity and Association Characters of Bacteria Isolated from Arbuscular Mycorrhizal Fungal Spore Walls

PubMed Central

Selvakumar, Gopal; Krishnamoorthy, Ramasamy; Kim, Kiyoon; Sa, Tong-Min

2016-01-01

Association between arbuscular mycorrhizal fungi (AMF) and bacteria has long been studied. However, the factors influencing their association in the natural environment is still unknown. This study aimed to isolate bacteria associated with spore walls of AMF and identify their potential characters for association. Spores collected from coastal reclamation land were differentiated based on their morphology and identified by 18S rDNA sequencing as Funneliformis caledonium, Racocetra alborosea and Funneliformis mosseae. Bacteria associated with AMF spore walls were isolated after treating them with disinfection solution at different time intervals. After 0, 10 and 20 min of spore disinfection, 86, 24 and 10 spore associated bacteria (SAB) were isolated, respectively. BOX-PCR fingerprinting analysis showed that diverse bacterial communities were associated to AMF spores. Bacteria belonging to the same genera could associate with different AMF spores. Gram positive bacteria were more closely associated with AMF spores. Isolated SAB were characterized and tested for spore association characters such as chitinase, protease, cellulase enzymes and exopolysaccharide production (EPS). Among the 120 SAB, 113 SAB were able to show one or more characters for association and seven SAB did not show any association characters. The 16S rDNA sequence of SAB revealed that bacteria belonging to the phyla Firmicutes, Proteobacteria, Actinobacteria and Bactereiodes were associated with AMF spore walls. PMID:27479250

Clostridium difficile shows no trade-off between toxin and spore production within the human host.

PubMed

Blanco, Natalia; Walk, Seth; Malani, Anurag N; Rickard, Alexander; Benn, Michele; Eisenberg, Marisa; Zhang, Min; Foxman, Betsy

2018-05-01

This study aimed to describe the correlation between Clostridium difficile spore and toxin levels within the human host. In addition, we assessed whether overgrowth of Candida albicans modified this association. We measured toxin, spore and Candida albicans levels among 200 successively collected stool samples that tested positive for C. difficile, and PCR ribotyped these C. difficile isolates. Analysis of variance and linear regression were used to test the association between spore and toxin levels. Kruskal-Wallis tests and t-tests were used to compare the association between spore or toxin levels and host, specimen, or pathogen characteristics. C. difficile toxin and spore levels were positively associated (P<0.001); this association did not vary significantly with C. albicans overgrowth [≥5 logs of C. albicans colony-forming units (c.f.u.) g -1 ]. However, ribotypes 027 and 078-126 were significantly associated with higher levels of toxin and spores, and C. albicans overgrowth. The strong positive association observed between in vivo levels of C. difficile toxin and spores suggests that patients with more severe C. difficile infections may have increased spore production, enhancing C. difficile transmission. Although, on average, spore levels were higher in toxin-positive samples than in toxin-negative/PCR-positive samples, spores were found in almost all toxin-negative samples. The ubiquity of spore production among toxin-negative and formed stool samples emphasizes the importance of following infection prevention and control measures for all C. difficile-positive patients during their entire hospital stay.

Correlation of spring spore concentrations and meteorological conditions in Tulsa, Oklahoma

NASA Astrophysics Data System (ADS)

Troutt, C.; Levetin, E.

Different spore types are abundant in the atmosphere depending on the weather conditions. Ascospores generally follow precipitation, while spore types such as Alternaria and Cladosporium are abundant in dry conditions. This project attempted to correlate fungal spore concentrations with meteorological data from Tulsa, Oklahoma during May 1998 and May 1999. Air samples were collected and analyzed by the 12-traverse method. The spore types included were Cladosporium, Alternaria, Epicoccum, Curvularia, Pithomyces, Drechslera, smut spores, ascospores, basidiospores, and other spores. Weather variables included precipitation levels, temperature, dew point, air pressure, wind speed, wind direction and wind gusts. There were over 242.57 mm of rainfall in May 1999 and only 64.01 mm in May 1998. The most abundant spore types during May 1998 and May 1999 were Cladosporium, ascospores, and basidiospores. Results showed that there were significant differences in the dry-air spora between May 1998 and May 1999. There were twice as many Cladosporium in May 1998 as in May 1999; both ascospores and basidiospores showed little change. Multiple regression analysis was used to determine which meteorological variables influenced spore concentrations. Results showed that there was no single model for all spore types. Different combinations of factors were predictors of concentration for the various fungi examined; however, temperature and dew point seemed to be the most important meteorological factors.

The sps Gene Products Affect the Germination, Hydrophobicity, and Protein Adsorption of Bacillus subtilis Spores

PubMed Central

Cangiano, Giuseppina; Sirec, Teja; Panarella, Cristina; Isticato, Rachele; Baccigalupi, Loredana; De Felice, Maurilio

2014-01-01

The multilayered surface of the Bacillus subtilis spore is composed of proteins and glycans. While over 70 different proteins have been identified as surface components, carbohydrates associated with the spore surface have not been characterized in detail yet. Bioinformatic data suggest that the 11 products of the sps operon are involved in the synthesis of polysaccharides present on the spore surface, but an experimental validation is available only for the four distal genes of the operon. Here, we report a transcriptional analysis of the sps operon and a functional study performed by constructing and analyzing two null mutants lacking either all or only the promoter-proximal gene of the operon. Our results show that both sps mutant spores apparently have normal coat and crust but have a small germination defect and are more hydrophobic than wild-type spores. We also show that spores lacking all Sps proteins are highly adhesive and form extensive clumps. In addition, sps mutant spores have an increased efficiency in adsorbing a heterologous enzyme, suggesting that hydrophobic force is a major determinant of spore adsorption and indicating that a deep understanding of the surface properties of the spore is essential for its full development as a surface display platform. PMID:25239894

Computer-assisted image processing to detect spores from the fungus Pandora neoaphidis.

PubMed

Korsnes, Reinert; Westrum, Karin; Fløistad, Erling; Klingen, Ingeborg

2016-01-01

This contribution demonstrates an example of experimental automatic image analysis to detect spores prepared on microscope slides derived from trapping. The application is to monitor aerial spore counts of the entomopathogenic fungus Pandora neoaphidis which may serve as a biological control agent for aphids. Automatic detection of such spores can therefore play a role in plant protection. The present approach for such detection is a modification of traditional manual microscopy of prepared slides, where autonomous image recording precedes computerised image analysis. The purpose of the present image analysis is to support human visual inspection of imagery data - not to replace it. The workflow has three components:•Preparation of slides for microscopy.•Image recording.•Computerised image processing where the initial part is, as usual, segmentation depending on the actual data product. Then comes identification of blobs, calculation of principal axes of blobs, symmetry operations and projection on a three parameter egg shape space.

Analysis of α-glucosidase enzyme activity used in a rapid test for steam sterilization assurance.

PubMed

Setlow, B; Korza, G; Setlow, P

2016-05-01

This study was to determine the sources, location and identity of α-glucosidases in dormant/germinating/outgrowing spores and growing cells of Geobacillus stearothermophilus ATCC 7953, an enzymatic activity in spores used in rapid tests of steam sterilization. α-Glucosidase activity in spores and cells was determined measuring methylumbelliferyl-α-d-glucoside (α-MUG) or α-MUG-6-phosphate hydrolysis fluorometrically. While α-MUG-6-phosphate was not hydrolysed by cell or spore extracts, assays with α-MUG showed that: (1) the α-glucosidase activity was inside and outside spores, and the activity outside spores was largely removed by buffer washes or heat activation, whereas α-glucosidase activity was only inside vegetative cells; (2) most α-glucosidase activity in cells and spores was soluble; (3) Western blots and enzyme inhibition using an anti-α-glucosidase antiserum identified ≥2 α-glucosidases in spores and growing cells; (4) α-glucosidase-specific activities were similar in dormant, germinated and outgrowing spore and growing cell extracts; and (5) significant α-glucosidase was synthesized during spore germination and outgrowth and cell growth, this synthesis was not repressed by glucose nor induced by α-MUG, but glucose inhibited α-MUG uptake. α-MUG hydrolysis by G. stearothermophilus is by α-MUG uptake and hydrolysis by ≥2 α-glucosidases associated with dormant spores and synthesized by germinating and outgrowing spores. The enzyme activity observed by sterilization assurance assays appears likely to come from heat-stable enzyme in the spore core and enzyme(s) synthesized in spore outgrowth. The results of this work provide new insight into the science behind a rapid test for steam sterilization assurance. © 2016 The Society for Applied Microbiology.

Comparative analysis of immune effects in mice model: Clonorchis sinensis cysteine protease generated from recombinant Escherichia coli and Bacillus subtilis spores.

PubMed

Wu, Zhanshuai; Tang, Zeli; Shang, Mei; Zhao, Lu; Zhou, Lina; Kong, Xiangzhan; Lin, Zhipeng; Sun, Hengchang; Chen, Tingjin; Xu, Jin; Li, Xuerong; Huang, Yan; Yu, Xinbing

2017-07-01

Clonorchiasis remains a nonnegligible public health problem in endemic areas. Cysteine protease of Clonorchis sinensis (CsCP) plays indispensable roles in the parasitic physiology and pathology, and has been exploited as a promising drug and vaccine candidate. In recent years, development of spore-based vaccines against multiple pathogens has attracted many investigators' interest. In previous studies, the recombinant Escherichia coli (BL21) and Bacillus subtilis spores expressing CsCP have been successfully constructed, respectively. In this study, the immune effects of CsCP protein purified from recombinant BL21 (rCsCP) and B. subtilis spores presenting CsCP (B.s-CsCP) in Balb/c mice model were conducted with comparative analysis. Levels of specific IgG, IgG1 and IgG2a were significantly increased in sera from both rCsCP and B.s-CsCP intraperitoneally immunized mice. Additionally, recombinant spores expressing abundant fusion CsCP (0.03125 pg/spore) could strongly enhance the immunogenicity of CsCP with significantly higher levels of IgG and isotypes. Compared with rCsCP alone, intraperitoneal administration of mice with spores expressing CsCP achieved a better effect of fighting against C. sinensis infection by slowing down the process of fibrosis. Our results demonstrated that a combination of Th1/Th2 immune responses could be elicited by rCsCP, while spores displaying CsCP prominently induced Th1-biased specific immune responses, and the complex cytokine network maybe mediates protective immune responses against C. sinensis. This work further confirmed that the usage of B. subtilis spores displaying CsCP is an effective way to against C. sinensis.

A Standard Method To Inactivate Bacillus anthracis Spores to Sterility via Gamma Irradiation

PubMed Central

Cote, Christopher K.; Buhr, Tony; Bernhards, Casey B.; Bohmke, Matthew D.; Calm, Alena M.; Esteban-Trexler, Josephine S.; Hunter, Melissa; Katoski, Sarah E.; Kennihan, Neil; Klimko, Christopher P.; Miller, Jeremy A.; Minter, Zachary A.; Pfarr, Jerry W.; Prugh, Amber M.; Quirk, Avery V.; Rivers, Bryan A.; Shea, April A.; Shoe, Jennifer L.; Sickler, Todd M.; Young, Alice A.; Fetterer, David P.; Welkos, Susan L.; McPherson, Derrell; Fountain, Augustus W.

2018-01-01

ABSTRACT In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 × 1010 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D10) values and irradiation doses required to ensure sterility (DSAL) to the point at which the probability of detecting a viable spore is 10−6. Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques. IMPORTANCE The inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism. PMID:29654186

An MSH4 homolog, stpp1, from Pleurotus pulmonarius is a "silver bullet" for resolving problems caused by spores in cultivated mushrooms.

PubMed

Okuda, Yasuhito; Murakami, Shigeyuki; Honda, Yoichi; Matsumoto, Teruyuki

2013-08-01

The enormous number of spores produced by fruiting bodies during cultivation of mushrooms can lead to allergic reactions of workers, reduction of commercial value, spread of mushroom disease, pollution of facilities, and depletion of genetic diversity in natural populations. A cultivar harboring a sporulation-deficient (sporeless) mutation would be very useful for preventing these problems, but sporeless commercial cultivars are very limited in usefulness because sporeless traits are often linked with traits that are unfavorable for commercial cultivation. Thus, identifying a causal gene of a sporeless phenotype not linked to the adverse traits in breeding and cultivation is crucial for the establishment of sporeless breeding using a strategy employing targeting induced local lesions in genomes (TILLING) in cultivated mushrooms. We used a Pleurotus pulmonarius (Fr.) Quél. sporeless strain to identify and characterize the single recessive gene controlling the mutation. The 3,853-bp stpp1 gene encodes a protein of 854 amino acids and belongs to the MutS homolog (MSH) family associated with mismatch repair in DNA synthesis or recombination in meiosis. Gene expression analysis of the fruiting body showed that this gene is strongly expressed in the gills. Phenotypic analysis of disruptants formed by gene targeting suggested a reproducible sporeless phenotype. Mutants deficient in a functional copy of this gene have no unfavorable traits for sporeless cultivar breeding, so this gene will be an extremely useful target for efficient and versatile sporeless breeding in P. pulmonarius and various other cultivated mushrooms.

Isolation and analysis of bacteria associated with spores of Gigaspora margarita.

PubMed

Cruz, A F; Horii, S; Ochiai, S; Yasuda, A; Ishii, T

2008-06-01

The aim of this work was to observe bacteria associated with the spores of Gigaspora margarita, an arbuscular mycorrhizal fungus (AMF). First, a direct analysis of DNA from sterilized spores indicated the bacteria belonging to the genus Janthinobacterium. In the second assay, two bacterial strains were isolated by osmosis from protoplasts, which were derived from spores by using two particular enzymes: lysing enzymes and yatalase. After isolation, cultivation and identification by their DNA as performed in the first experiment, the species with the closest relation were Janthinobacterium lividum (KCIGM01) and Paenibacillus polymyxa (KCIGM04) isolated with lysing enzymes and yatalase respectively. Morphologically, J. lividum was Gram negative and oval, while P. polymyxa was also oval, but Gram positive. Both strains had antagonistic effects to the pathogenic fungi Rosellimia necatrix, Pythium ultimum, Fusarium oxysporum and Rhizoctonia solani. In particular, J. lividum was much stronger in this role. However, in phosphorus (P) solubilization P. polymyxa functioned better than J. lividum. This experiment had revealed two new bacteria species (P. polymyxa and J. lividum), associated with AMF spores, which functioned to suppress diseases and to solubilize P. AMF spores could be a useful source for bacterial antagonists to soil-borne diseases and P solubilization.

Measurement and analysis on optical characteristics of Aspergillus oryzae spores in infrared band

NASA Astrophysics Data System (ADS)

Li, Le; Hu, Yihua; Gu, Youlin; Chen, Wei; Xu, Shilong; Zhao, Xinying

2015-10-01

Spore is an important part of bioaerosols. The optical characteristics of spore is a crucial parameter for study on bioaerosols. The reflection within the waveband of 2.5 to15μm were measured by squash method. Based on the measured data, Complex refractive index of Aspergillus oryzae spores within the waveband of 3 to 5μm and 8 to 14 μm were calculated by using Krames-Kronig (K-K) relationship. Then,the mass extinction coefficient of Aspergillus oryzae spores within the waveband of 3 to 5μm and 8 to 14μm were obtained by utilizing Mie scattering theory, and the results were analyzed and discussed. The average mass extinction coefficient of Aspergillus oryzae spores is 0.51 m2/g in the range of 3 to 5μm and 0.48m2/g in the range of 8 to 14μm. Compared with common inorganic compounds, Aspergillus oryzae spores possesses a good extinction performance in infrared band.

A novel live attenuated anthrax spore vaccine based on an acapsular Bacillus anthracis Sterne strain with mutations in the htrA, lef and cya genes.

PubMed

Chitlaru, Theodor; Israeli, Ma'ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Ehrlich, Sharon; Cohen, Ofer; Shafferman, Avigdor

2017-10-20

We recently reported the development of a novel, next-generation, live attenuated anthrax spore vaccine based on disruption of the htrA (High Temperature Requirement A) gene in the Bacillus anthracis Sterne veterinary vaccine strain. This vaccine exhibited a highly significant decrease in virulence in murine, guinea pig and rabbit animal models yet preserved the protective value of the parental Sterne strain. Here, we report the evaluation of additional mutations in the lef and cya genes, encoding for the toxin components lethal factor (LF) and edema factor (EF), to further attenuate the SterneΔhtrA strain and improve its compatibility for human use. Accordingly, we constructed seven B. anthracis Sterne-derived strains exhibiting different combinations of mutations in the htrA, cya and lef genes. The various strains were indistinguishable in growth in vitro and in their ability to synthesise the protective antigen (PA, necessary for the elicitation of protection). In the sensitive murine model, we observed a gradual increase (ΔhtrA<ΔhtrAΔcya<ΔhtrAΔlef<ΔhtrAΔlefΔcya) in attenuation - up to 10 8 -fold relative to the parental Sterne vaccine strain. Most importantly, all various SterneΔhtrA derivative strains did not differ in their ability to elicit protective immunity in guinea pigs. Immunisation of guinea pigs with a single dose (10 9 spores) or double doses (>10 7 spores) of the most attenuated triple mutant strain SterneΔhtrAlef MUT Δcya induced a robust immune response, providing complete protection against a subsequent respiratory lethal challenge. Partial protection was observed in animals vaccinated with a double dose of as few as 10 5 spores. Furthermore, protective immune status was maintained in all vaccinated guinea pigs and rabbits for at least 40 and 30weeks, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analysis of the Spore Membrane Proteome in Clostridium perfringens Implicates Cyanophycin in Spore Assembly.

PubMed

Liu, Hualan; Ray, W Keith; Helm, Richard F; Popham, David L; Melville, Stephen B

2016-06-15

Heat-resistant endospore formation plays an important role in Clostridium perfringens-associated foodborne illnesses. The spores allow the bacterium to survive heating during normal cooking processes, followed by germination and outgrowth of the bacterium in contaminated foods. To identify proteins associated with germination and other spore functions, a comparative spore membrane proteome analysis of dormant and germinated spores of C. perfringens strain SM101 was performed by using gel-based protein separation and liquid chromatography coupled with matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) mass spectrometry. A total of 494 proteins were identified, and 117 of them were predicted to be integral membrane or membrane-associated proteins. Among these membrane proteins, 16 and 26 were detected only in dormant and germinated spores, respectively. One protein that was detected only in germinated spore membranes was the enzyme cyanophycinase, a protease that cleaves the polymer cyanophycin, which is composed of l-arginine-poly(l-aspartic acid), to β-Asp-Arg. Genes encoding cyanophycinase and cyanophycin synthetase have been observed in many species of Clostridium, but their role has not been defined. To determine the function of cyanophycin in C. perfringens, a mutation was introduced into the cphA gene, encoding cyanophycin synthetase. In comparison to parent strain SM101, the spores of the mutant strain retained wild-type levels of heat resistance, but fewer spores were made, and they were smaller, suggesting that cyanophycin synthesis plays a role in spore assembly. Although cyanophycin could not be extracted from sporulating C. perfringens cells, an Escherichia coli strain expressing the cphA gene made copious amounts of cyanophycin, confirming that cphA encodes a cyanophycin synthetase. Clostridium perfringens is a common cause of food poisoning, and germination of spores after cooking is thought to play a significant role in the disease. How C. perfringens controls the germination process is still not completely understood. We characterized the proteome of the membranes from dormant and germinated spores and discovered that large-scale changes occur after germination is initiated. One of the proteins that was detected after germination was the enzyme cyanophycinase, which degrades the storage compound cyanophycin, which is found in cyanobacteria and other prokaryotes. A cyanophycin synthetase mutant was constructed and found to make spores with altered morphology but normal heat resistance, suggesting that cyanophycin plays a different role in C. perfringens than it does in cyanobacteria. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amidan, Brett G.; Hutchison, Janine R.

There are many sources of variability that exist in the sample collection and analysis process. This paper addresses many, but not all, sources of variability. The main focus of this paper was to better understand and estimate variability due to differences between samplers. Variability between days was also studied, as well as random variability within each sampler. Experiments were performed using multiple surface materials (ceramic and stainless steel), multiple contaminant concentrations (10 spores and 100 spores), and with and without the presence of interfering material. All testing was done with sponge sticks using 10-inch by 10-inch coupons. Bacillus atrophaeus wasmore » used as the BA surrogate. Spores were deposited using wet deposition. Grime was coated on the coupons which were planned to include the interfering material (Section 3.3). Samples were prepared and analyzed at PNNL using CDC protocol (Section 3.4) and then cultured and counted. Five samplers were trained so that samples were taken using the same protocol. Each sampler randomly sampled eight coupons each day, four coupons with 10 spores deposited and four coupons with 100 spores deposited. Each day consisted of one material being tested. The clean samples (no interfering materials) were run first, followed by the dirty samples (coated with interfering material). There was a significant difference in recovery efficiency between the coupons with 10 spores deposited (mean of 48.9%) and those with 100 spores deposited (mean of 59.8%). There was no general significant difference between the clean and dirty (containing interfering material) coupons or between the two surface materials; however, there was a significant interaction between concentration amount and presence of interfering material. The recovery efficiency was close to the same for coupons with 10 spores deposited, but for the coupons with 100 spores deposited, the recovery efficiency for the dirty samples was significantly larger (65.9% - dirty vs. 53.6% - clean) (see Figure 4.1). Variance component analysis was used to estimate the amount of variability for each source of variability. There wasn’t much difference in variability for dirty and clean samples, as well as between materials, so these results were pooled together. There was a significant difference in amount of concentration deposited, so results were separated for the 10 spore and 100 spore deposited tests. In each case the within sampler variability was the largest with variances of 426.2 for 10 spores and 173.1 for 100 spores. The within sampler variability constitutes the variability between the four samples of similar material, interfering material, and concentration taken by each sampler. The between sampler variance was estimated to be 0 for 10 spores and 1.2 for 100 spores. The between day variance was estimated to be 42.1 for 10 spores and 78.9 for 100 spores. Standard deviations can be calculated in each case by taking the square root of the variance.« less

The Molecular Timeline of a Reviving Bacterial Spore

PubMed Central

Sinai, Lior; Rosenberg, Alex; Smith, Yoav; Segev, Einat; Ben-Yehuda, Sigal

2015-01-01

Summary The bacterial spore can rapidly convert from a dormant to a fully active cell. Here we study this remarkable cellular transition in Bacillus subtilis and reveal the identity of the newly synthesized proteins throughout spore revival. Our analysis uncovers a highly ordered developmental program that correlates with the spore morphological changes and reveals the spatial and temporal molecular events fundamental to reconstruct a cell. As opposed to current knowledge, we found that translation takes place during the earliest revival event, termed germination, a process hitherto considered to occur without the need for any macromolecule synthesis. Furthermore, we demonstrate that translation is required for execution of germination and relies on the bona fide translational factors RpmE and Tig. Our study sheds light on the spore revival process and on the vital building blocks underlying cellular awakening, thereby paving the way for designing new antimicrobial agents to eradicate spore-forming pathogens. PMID:25661487

Residual Agar Determination in Bacterial Spores by Electrospray Ionization Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wahl, Karen L.; Colburn, Heather A.; Wunschel, David S.

2010-02-15

Presented here is an analytical method to detect residual agar from a bacterial spore sample as an indication of culturing on an agar plate. This method is based on the resolubilization of agar polysaccharide from a bacterial spore sample, enzymatic digestion, followed by electrospray ionization tandem mass spectrometry (ESI-MSn) analysis for detection of a specific agar fragment ion. A range of Bacillus species and strains were selected to demonstrate the effectiveness of this approach. The characteristic agar fragment ion was detected in the spores grown on agar that were washed from 1 to 5 times, irradiated or non-irradiated and notmore » in the spores grown in broth. A sample containing approximately 108 spores is currently needed for confident detection of residual agar from culture on agar plates in the presence of bacterial spores with a limit of detection of approximately 1 ppm agar spiked into a broth-grown spore sample. The results of a proficiency test with 42 blinded samples are presented demonstrating the utility of this method with no false positives and only 3 false negatives for samples that were below the detection level of the method as documented.« less

Role of mechanical vs. chemical action in the removal of adherent Bacillus spores during CIP procedures.

PubMed

Faille, C; Bénézech, T; Blel, W; Ronse, A; Ronse, G; Clarisse, M; Slomianny, C

2013-04-01

This study was designed to evaluate the respective roles of mechanical and chemical effects on the removal of Bacillus spores during cleaning-in-place. This analysis was performed on 12 strains belonging to the Bacillus cereus group (B. cereus, Bacillus anthracis, Bacillus thuringiensis) or to less related Bacillus species (Bacillus pumilus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus subtilis). Adherent spores were subjected to rinsing-in-place (mechanical action) and cleaning-in-place (mechanical and chemical actions) procedures, the latter involving NaOH 0.5% at 60°C. Results revealed that mechanical action alone only removed between 53 and 89% of the attached spores at a shear stress of 500 Pa. This resistance to shear was not related to spore surface properties. Conversely, in the presence of NaOH at a shear stress of 4 Pa, spores were readily detached, with between 80 and 99% of the adherent spores detached during CIP and the chemical action greatly depended on the strain. This finding suggests that chemical action plays the major role during CIP, whose efficacy is significantly governed by the spore surface chemistry. Copyright © 2012 Elsevier Ltd. All rights reserved.

Inhibitive effect on apoptosis in splenic lymphocytes of mice pretreated with lingzhi (Ganoderma lucidum) spores.

PubMed

Wang, Quanxi; Huang, Yifan; Wu, Baocheng; Mei, Jingliang; Zhang, Honglei; Qi, Baomin

2014-04-01

To investigate how the pretreatment of mice with Ganoderma spores affected the apoptosis of their splenic lymphocytes induced by dexamethasone after 19 days treatment. Sixty Kunming mice were randomly divided into six groups: blank control groupdrenched with normal saline; a drug control group drenched with 150 mg/mL Ganoderma spores; a model group treated with saline; a low dose group with 50 mg/mL Ganoderma spores; a moderate dose group with 100 mg/mL Ganoderma spores; and a high dose group with 150 mg/mL Ganoderma spores. The effect of Ganoderma spores on apoptosis in spleen lymphocytes was analyzed. All groups were treated for 19 days. On day 20, the model group and the 3 treatment groups were intraperitoneally injected dexamethasone to induce apoptosis. Splenic index and apoptosis indes were employed to measure cell apoptosis. The results showed that Ganoderma spores reduced the splenic index to different degrees in each group and the best effect was seen in the high dose group (P < 0.05).Terminal dexynucleotidyl transferase (TdT)-mediated 2'-Deoxyuridine 5'-Triphosphate nick end labeling staining revealed that the apoptotic index in all groups administered Ganoderma spores differed significantly from the model group, and a dose-response was observed. Flow cytometric analysis indicated that spleen lymphocyte apoptosis in the model group was extensive. Each dose of Ganoderma spores inhibited dexamethasone-induced apoptosis in spleen lymphocytes, and a dose-response was observed as well. The highest dose of Ganoderma spores decreased Malondialdehyde content in serum induced by dexamethasone (P < 0.05). The findings imply that the pretreatment of the mice with Ganoderma spores could reduce the apoptosis rate induced by dexamethasone in their splenic lymphocytes.

Atmospheric Pressure-Thermal Desorption (AP-TD)/Electrospray Ionization-Mass Spectrometry for the Rapid Analysis of Bacillus Spores

USDA-ARS?s Scientific Manuscript database

A technique is described where an atmospheric pressure-thermal desorption (AP-TD) device and electrospray ionization (ESI)-mass spectrometry are coupled and used for the rapid analysis of Bacillus spores in complex matrices. The resulting AP-TD/ESI-MS technique combines the generation of volatile co...

RNA-seq analysis identifies potential modulators of gravity response in spores of Ceratopteris (Parkeriaceae): evidence for modulation by calcium pumps and apyrase activity.

PubMed

Bushart, Thomas J; Cannon, Ashley E; Ul Haque, Aeraj; San Miguel, Phillip; Mostajeran, Kathy; Clark, Gregory B; Porterfield, D Marshall; Roux, Stanley J

2013-01-01

Gravity regulates the magnitude and direction of a trans-cell calcium current in germinating spores of Ceratopteris richardii. Blocking this current with nifedipine blocks the spore's downward polarity alignment, a polarization that is fixed by gravity ∼10 h after light induces the spores to germinate. RNA-seq analysis at 10 h was used to identify genes potentially important for the gravity response. The data set will be valuable for other developmental and phylogenetic studies. De novo Newbler assembly of 958 527 reads from Roche 454 sequencing was executed. The sequences were identified and analyzed using in silico methods. The roles of endomembrane Ca(2+)-ATPase pumps and apyrases in the gravity response were further tested using pharmacological agents. Transcripts related to calcium signaling and ethylene biosynthesis were identified as notable constituents of the transcriptome. Inhibiting the activity of endomembrane Ca(2+)-ATPase pumps with 2,5-di-(t-butyl)-1,4-hydroquinone diminished the trans-cell current, but increased the orientation of the polar axis to gravity. The effects of applied nucleotides and purinoceptor antagonists gave novel evidence implicating extracellular nucleotides as regulators of the gravity response in these fern spores. In addition to revealing general features of the transcriptome of germinating spores, the results highlight a number of calcium-responsive and light-receptive transcripts. Pharmacologic assays indicate endomembrane Ca(2+)-ATPases and extracellular nucleotides may play regulatory roles in the gravity response of Ceratopteris spores.

A microfluidic device for real-time monitoring of Bacillus subtilis bacterial spores during germination based on non-specific physicochemical interactions on the nanoscale level.

PubMed

Zabrocka, L; Langer, K; Michalski, A; Kocik, J; Langer, J J

2015-01-07

A microfluidic device for studies on the germination of bacterial spores (e.g. Bacillus subtilis) based on non-specific interactions on the nanoscale is presented. A decrease in the population of spores during germination followed by the appearance of transition forms and an increase in the number of vegetative cells can be registered directly and simultaneously by using the microfluidic device, which is equipped with a conductive polymer layer (polyaniline) in the form of a nano-network. The lab-on-a-chip-type device, operating in a continuous flow regime, allows monitoring of germination of bacterial spores and analysis of the process in detail. The procedure is fast and accurate enough for quantitative real-time monitoring of the main steps of germination, including final transformation of the spores into vegetative cells. All of this is done without the use of biomarkers or any bio-specific materials, such as enzymes, antibodies and aptamers, and is simply based on an analysis of physicochemical interactions on the nanoscale level.

The Bacillus subtilis yaaH Gene Is Transcribed by SigE RNA Polymerase during Sporulation, and Its Product Is Involved in Germination of Spores

PubMed Central

Kodama, Takeko; Takamatsu, Hiromu; Asai, Kei; Kobayashi, Kazuo; Ogasawara, Naotake; Watabe, Kazuhito

1999-01-01

The expression of 21 novel genes located in the region from dnaA to abrB of the Bacillus subtilis chromosome was analyzed. One of the genes, yaaH, had a predicted promoter sequence conserved among SigE-dependent genes. Northern blot analysis revealed that yaaH mRNA was first detected from 2 h after the cessation of logarithmic growth (T2) of sporulation in wild-type cells and in spoIIIG (SigG−) and spoIVCB (SigK−) mutants but not in spoIIAC (SigF−) and spoIIGAB (SigE−) mutants. The transcription start point was determined by primer extension analysis; the −10 and −35 regions are very similar to the consensus sequences recognized by SigE-containing RNA polymerase. A YaaH-His tag fusion encoded by a plasmid with a predicted promoter for the yaaH gene was produced from T2 of sporulation in a B. subtilis transformant and extracted from mature spores, indicating that the yaaH gene product is a spore protein. Inactivation of the yaaH gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yaaH mutant spores in a mixture of l-asparagine, d-glucose, d-fructose, and potassium chloride was almost the same as that of wild-type spores, but the mutant spores were defective in l-alanine-stimulated germination. These results suggest that yaaH is a novel gene encoding a spore protein produced in the mother cell compartment from T2 of sporulation and that it is required for the l-alanine-stimulated germination pathway. PMID:10419957

A novel gene: sawD related to the differentiation of streptomyces ansochromogenes.

PubMed

Gang, L; Wei, C; Yuqing, T; Huarong, T; Chater, K F; Buttner, M J

1999-01-01

A 1.3 kb DNA fragment was cloned from a total DNA library of Streptomyces ansochromogenes using Southern hybridization. Nucleotide sequencing analysis indicated that the 1320 bp DNA fragment contained a complete open reading frame (ORF). In search of databases, the deduced product of ORF containing 213 amino acids is homologous to the serine protease of Caulobacter cresceatus, and a conserved serine-catalytic active site (GPSAG) exists. The gene was designated as sawD. The function of this gene was studied with the strategy of gene disruption, and the result showed that the sawD may be related to sporulation and especially to the spore septation in Streptomyces ansochromogenes. The preliminary result indicated that sawD mutant could produce abundant pigment in contrast with the wild type, it seems that sawD gene may be involved in pigment biosynthesis, and this gene is also dispensable for biosynthesis of nikkomycin in Streptomyces ansochromogenes.

The pyruvate kinase of Stigmatella aurantiaca is an indole binding protein and essential for development.

PubMed

Stamm, Irmela; Lottspeich, Friedrich; Plaga, Wulf

2005-06-01

Myxospore formation of the myxobacterium Stigmatella aurantiaca can be uncoupled from the cooperative development i.e. fruiting body formation, by low concentrations of indole. Two putative indole receptor proteins were isolated by their capacity to bind indole and identified as pyruvate kinase (PK) and aldehyde dehydrogenase. The PK activity of Stigmatella crude extracts was stimulated by indole. Cloning of the PK gene (pykA) and the construction of a pykA disruption mutant strikingly revealed that PK is essential for multicellular development: Fruiting body formation was abolished in the mutant strain and indole-induced spore formation was delayed. The developmental defects could be complemented by insertion of the pykA gene at the mtaB locus of the Stigmatella genome excluding any polar effects of the pykA disruption.

NanoSIMS analysis of Bacillus spores for forensics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weber, P K; Davisson, M L; Velsko, S P

2010-02-23

The threat associated with the potential use of radiological, nuclear, chemical and biological materials in terrorist acts has resulted in new fields of forensic science requiring the application of state-of-the-science analytical techniques. Since the anthrax letter attacks in the United States in the fall of 2001, there has been increased interest in physical and chemical characterization of bacterial spores. While molecular methods are powerful tools for identifying genetic differences, other methods may be able to differentiate genetically identical samples based on physical and chemical properties, as well as provide complimentary information, such as methods of production and approximate date ofmore » production. Microanalysis has the potential to contribute significantly to microbial forensics. Bacillus spores are highly structured, consisting of a core, cortex, coat, and in some species, an exosporium. This structure provides a template for constraining elemental abundance differences at the nanometer scale. The primary controls on the distribution of major elements in spores are likely structural and physiological. For example, P and Ca are known to be abundant in the spore core because that is where P-rich nucleic acids and Cadipicolinic acid are located, respectively. Trace elements are known to bind to the spore coat but the controls on these elements are less well understood. Elemental distributions and abundances may be directly related to spore production, purification and stabilization methodologies, which are of particular interest for forensic investigation. To this end, we are developing a high-resolution secondary ion mass spectrometry method using a Cameca NanoSIMS 50 to study the distribution and abundance of trace elements in bacterial spores. In this presentation we will review and compare methods for preparing and analyzing samples, as well as review results on the distribution and abundance of elements in bacterial spores. We use NanoSIMS to directly image samples as well as depth profile samples. The directly imaged samples are sectioned to present a flat surface for analysis. We use focused ion beam (FIB) milling to top-cut individual spores to create flat surfaces for NanoSIMS analysis. Depth profiling can be used on whole spores, which are consumed in the process of analysis. The two methods generate comparable results, with the expected distribution of P and Ca. Ca-compatible elements, such as Mg and Mn, are found to follow the distribution of Ca. The distribution of other elements will be discussed. We envision the first application of this methodology will be to sample matching for trace samples. Towards this end, we are generating a baseline data set for samples produced by multiple laboratories. Preliminary results suggest that this method provides significant probative value for identifying samples produced by the same method in the same laboratory, as well as coming from the same initial production run. The results of this study will be presented.« less

Back-trajectory modelling and DNA-based species-specific detection methods allow tracking of fungal spore transport in air masses.

PubMed

Grinn-Gofroń, Agnieszka; Sadyś, Magdalena; Kaczmarek, Joanna; Bednarz, Aleksandra; Pawłowska, Sylwia; Jedryczka, Malgorzata

2016-11-15

Recent advances in molecular detection of living organisms facilitate the introduction of novel methods to studies of the transport of fungal spores over large distances. Monitoring the migration of airborne fungi using microscope based spore identification is limited when different species produce very similar spores. In our study, DNA-based monitoring with the use of species-specific probes allowed us to track the aerial movements of two important fungal pathogens of oilseed rape (Brassica napus L.), i.e., Leptosphaeria maculans and Leptosphaeria biglobosa, which have identical spore shape and size. The fungi were identified using dual-labelled fluorescent probes that were targeted to a β-tubulin gene fragment of either Leptosphaeria species. Spore identification by Real-Time PCR techniques capable of detecting minute amounts of DNA of selected fungal species was combined with back-trajectory analysis, allowing the tracking of past movements of air masses using the Hybrid Single Particle Lagrangian Integrated Trajectory model. Over a study period spanning the previous decade (2006-2015) we investigated two specific events relating to the long distance transport of Leptosphaeria spp. spores to Szczecin in North-West Poland. Based on the above mentioned methods and the results obtained with the additional spore sampler located in nearby Szczecin, and operating at the ground level in an oilseed rape field, we have demonstrated that on both occasions the L. biglobosa spores originated from the Jutland Peninsula. This is the first successful attempt to combine analysis of back-trajectories of air masses with DNA-based identification of economically important pathogens of oilseed rape in Europe. In our studies, the timing of L. biglobosa ascospore dispersal in the air was unlikely to result in the infection of winter oilseed rape grown as a crop plant. However, the fungus could infect other host plants, such as vegetable brassicas, cruciferous weeds, spring rapeseed and winter rapeseed growing as a volunteer plant. Copyright © 2016 Elsevier B.V. All rights reserved.

Conserved Oligopeptide Permeases Modulate Sporulation Initiation in Clostridium difficile

PubMed Central

Edwards, Adrianne N.; Nawrocki, Kathryn L.

2014-01-01

The anaerobic gastrointestinal pathogen Clostridium difficile must form a metabolically dormant spore to survive in oxygenic environments and be transmitted from host to host. The regulatory factors by which C. difficile initiates and controls the early stages of sporulation in C. difficile are not highly conserved in other Clostridium or Bacillus species. Here, we investigated the role of two conserved oligopeptide permeases, Opp and App, in the regulation of sporulation in C. difficile. These permeases are known to positively affect sporulation in Bacillus species through the import of sporulation-specific quorum-sensing peptides. In contrast to other spore-forming bacteria, we discovered that inactivating these permeases in C. difficile resulted in the earlier expression of early sporulation genes and increased sporulation in vitro. Furthermore, disruption of opp and app resulted in greater virulence and increased the amounts of spores recovered from feces in the hamster model of C. difficile infection. Our data suggest that Opp and App indirectly inhibit sporulation, likely through the activities of the transcriptional regulator SinR and its inhibitor, SinI. Taken together, these results indicate that the Opp and App transporters serve a different function in controlling sporulation and virulence in C. difficile than in Bacillus subtilis and suggest that nutrient availability plays a significant role in pathogenesis and sporulation in vivo. This study suggests a link between the nutritional status of the environment and sporulation initiation in C. difficile. PMID:25069979

Proteins encoded by the gerP operon are localised to the inner coat in Bacilluscereus spores and are dependent on GerPA and SafA for assembly.

PubMed

Ghosh, Abhinaba; Manton, James D; Mustafa, Amin R; Gupta, Mudit; Ayuso-Garcia, Alejandro; Rees, Eric J; Christie, Graham

2018-05-04

Germination of Bacillus spores is triggered by certain amino acids and sugar molecules, which permeate through the outermost layers of the spore to interact with receptor complexes that reside in the inner membrane. Previous studies have shown that mutations in the hexacistronic gerP locus reduce the rate of spore germination, with experimental evidence indicating that the defect stems from reduced permeability of the spore coat to germinant molecules. Here we use the ellipsoid localisation microscopy technique to reveal that all six Bacillus cereus GerP proteins share proximity with cortex lytic enzymes within the inner coat. We reveal also that the GerPA protein alone can localise in the absence of all other GerP proteins, and that it has an essential role for the localisation of all other GerP proteins within the spore. The latter is also demonstrated to be SafA - but not CotE - dependent for localisation, which is consistent with an inner coat location. GerP null spores are shown also to have reduced permeability to fluorescently labelled dextran molecules compared to wild type spores. Overall, the results support the hypothesis that the GerP proteins have a structural role within the spore associated with coat permeability. Importance The bacterial spore coat comprises a multi-layered proteinaceous structure that influences the distribution, survival and germination properties of spores in the environment. Results from the current study are significant since they increase our understanding of coat assembly and architecture while adding detail to existing models of germination. We demonstrate also that the ELM image analysis technique can be used as a novel tool to provide direct quantitative measurements of spore coat permeability. Progress in all of these areas should ultimately facilitate improved methods of spore control in a range of industrial, healthcare and environmental sectors. Copyright © 2018 American Society for Microbiology.

Relationships between airborne fungal spore concentration of Cladosporium and the summer climate at two sites in Britain

NASA Astrophysics Data System (ADS)

Hollins, P. D.; Kettlewell, P. S.; Atkinson, M. D.; Stephenson, D. B.; Corden, J. M.; Millington, W. M.; Mullins, J.

Cladosporium conidia have been shown to be important aeroallergens in many regions throughout the world, but annual spore concentrations vary considerably between years. Understanding these annual fluctuations may be of value in the clinical management of allergies. This study investigates the number of days in summer when spore concentration exceeds the allergenic threshold in relation to regional temperature and precipitation at two sites in England and Wales over 27 years. Results indicate that number of days in summer when the Cladosporium spores are above the allergenic concentration is positively correlated with regional temperature and negatively correlated with precipitation for both sites over the study period. Further analysis used a winter North Atlantic Oscillation index to explore the potential for long-range forecasting of the aeroallergen. For both spore measurement sites, a positive correlation exists between the winter North Atlantic Oscillation index and the number of days in summer above the allergenic threshold for Cladosporium spore concentration.

Robust Phagocyte Recruitment Controls the Opportunistic Fungal Pathogen Mucor circinelloides in Innate Granulomas In Vivo.

PubMed

Inglesfield, Sarah; Jasiulewicz, Aleksandra; Hopwood, Matthew; Tyrrell, James; Youlden, George; Mazon-Moya, Maria; Millington, Owain R; Mostowy, Serge; Jabbari, Sara; Voelz, Kerstin

2018-03-27

Mucormycosis is an emerging fungal infection with extremely high mortality rates in patients with defects in their innate immune response, specifically in functions mediated through phagocytes. However, we currently have a limited understanding of the molecular and cellular interactions between these innate immune effectors and mucormycete spores during the early immune response. Here, the early events of innate immune recruitment in response to infection by Mucor circinelloides spores are modeled by a combined in silico modeling approach and real-time in vivo microscopy. Phagocytes are rapidly recruited to the site of infection in a zebrafish larval model of mucormycosis. This robust early recruitment protects from disease onset in vivo In silico analysis identified that protection is dependent on the number of phagocytes at the infection site, but not the speed of recruitment. The mathematical model highlights the role of proinflammatory signals for phagocyte recruitment and the importance of inhibition of spore germination for protection from active fungal disease. These in silico data are supported by an in vivo lack of fungal spore killing and lack of reactive oxygen burst, which together result in latent fungal infection. During this latent stage of infection, spores are controlled in innate granulomas in vivo Disease can be reactivated by immunosuppression. Together, these data represent the first in vivo real-time analysis of innate granuloma formation during the early stages of a fungal infection. The results highlight a potential latent stage during mucormycosis that should urgently be considered for clinical management of patients. IMPORTANCE Mucormycosis is a dramatic fungal infection frequently leading to the death of patients. We know little about the immune response to the fungus causing this infection, although evidence points toward defects in early immune events after infection. Here, we dissect this early immune response to infectious fungal spores. We show that specialized white blood cells (phagocytes) rapidly respond to these spores and accumulate around the fungus. However, we demonstrate that the mechanisms that enable phagocytes to kill the fungus fail, allowing for survival of spores. Instead a cluster of phagocytes resembling an early granuloma is formed around spores to control the latent infection. This study is the first detailed analysis of early granuloma formation during a fungal infection highlighting a latent stage that needs to be considered for clinical management of patients. Copyright © 2018 Inglesfield et al.

Engineered strains of Streptococcus macedonicus towards an osmotic stress resistant phenotype retain their ability to produce the bacteriocin macedocin under hyperosmotic conditions.

PubMed

Anastasiou, Rania; Driessche, Gonzalez Van; Boutou, Effrossyni; Kazou, Maria; Alexandraki, Voula; Vorgias, Constantinos E; Devreese, Bart; Tsakalidou, Effie; Papadimitriou, Konstantinos

2015-10-20

Streptococcus macedonicus ACA-DC 198 produces the bacteriocin macedocin in milk only under low NaCl concentrations (<1.0%w/v). The thermosensitive plasmid pGh9:ISS1 was employed to generate osmotic stress resistant (osmr) mutants of S. macedonicus. Three osmr mutants showing integration of the vector in unique chromosomal sites were identified and the disrupted loci were characterized. Interestingly, the mutants were able to grow and to produce macedocin at considerably higher concentrations of NaCl compared to the wild-type (up to 4.0%w/v). The production of macedocin under hyperosmotic conditions solely by the osmr mutants was validated by the well diffusion assay and by mass spectrometry analysis. RT-PCR experiments demonstrated that the macedocin biosynthetic regulon was transcribed at high salt concentrations only in the mutants. Mutant osmr3, the most robust mutant, was converted in its markerless derivative (osmr3f). Co-culture of S. macedonicus with spores of Clostridium tyrobutyricum in milk demonstrated that only the osmr3f mutant and not the wild-type inhibited the growth of the spores under hyperosmotic conditions (i.e., 2.5%w/v NaCl) due to the production of macedocin. Our study shows how genetic manipulation of a strain towards a stress resistant phenotype could improve bacteriocin production under conditions of the same stress. Copyright © 2015 Elsevier B.V. All rights reserved.

Low-Temperature Sterilization with Surface-Wave-Excited Oxygen Plasma

NASA Astrophysics Data System (ADS)

Nagatsu, Masaaki; Terashita, Fumie; Koide, Yukio

2003-07-01

Low-temperature plasma sterilization has been experimentally demonstrated using surface-wave plasma excited by a 2.45 GHz microwave. With the spores of Bacillus stearothermophilus and Bacillus subtilis as biological indicators, we have carried out the plasma sterilization experiments by varying the irradiation period of oxygen plasma discharges. It was experimentally confirmed that the spores with a population of 1.5 × 106 were sterilized by irradiating them with oxygen plasma discharges generated with a microwave power of 700 W at a pressure of 60-80 mTorr for 3 min or longer. From the scanning electron microscopy (SEM) analysis of the spores, we found that the sterilized spores clearly had different sizes and shapes compared with those before the plasma irradiation. Furthermore, present experiments suggested that the changes of spore shapes were mainly attributed to the reactive interactions with oxygen radicals.

Spores of the mycorrhizal fungus Glomus mosseae host yeasts that solubilize phosphate and accumulate polyphosphates.

PubMed

Mirabal Alonso, Loreli; Kleiner, Diethelm; Ortega, Eduardo

2008-04-01

The present paper reports the presence of bacteria and yeasts tightly associated with spores of an isolate of Glomus mosseae. Healthy spores were surface disinfected by combining chloramine-T 5%, Tween-40, and cephalexin 2.5 g L(-1) (CTCf). Macerates of these spores were incubated on agar media, microorganisms were isolated, and two yeasts were characterized (EndoGm1, EndoGm11). Both yeasts were able to solubilize low-soluble P sources (Ca and Fe phosphates) and accumulate polyphosphates (polyPs). Sequence analysis of 18S ribosomal deoxyribonucleic acid showed that the yeasts belong to the genera Rhodotorula or Rhodosporidium (EndoGm1) and Cryptococcus (EndoGm11). Results from inoculation experiments showed an effect of the spore-associated yeasts on the root growth of rice, suggesting potential tripartite interactions with mycorrhizal fungi and plants.

The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis.

PubMed

Wang, Katherine H; Isidro, Anabela L; Domingues, Lia; Eskandarian, Haig A; McKenney, Peter T; Drew, Kevin; Grabowski, Paul; Chua, Ming-Hsiu; Barry, Samantha N; Guan, Michelle; Bonneau, Richard; Henriques, Adriano O; Eichenberger, Patrick

2009-11-01

Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to green fluorescent protein and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homologue of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement.

The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis

PubMed Central

Wang, Katherine H.; Isidro, Anabela L.; Domingues, Lia; Eskandarian, Haig A.; McKenney, Peter T.; Drew, Kevin; Grabowski, Paul; Chua, Ming-Hsiu; Barry, Samantha N.; Guan, Michelle; Bonneau, Richard; Henriques, Adriano O.; Eichenberger, Patrick

2009-01-01

SUMMARY Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to GFP and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homolog of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement. PMID:19775244

The effects of meteorological factors on the occurrence of Ganoderma sp. spores in the air

NASA Astrophysics Data System (ADS)

Grinn-Gofroń, Agnieszka; Strzelczak, Agnieszka

2011-03-01

Ganoderma sp. is an airborne fungal spore type known to trigger respiratory allergy symptoms in sensitive patients. Aiming to reduce the risk for allergic individuals, we analysed fungal spore circulation in Szczecin, Poland, and its dependence on meteorological conditions. Statistical models for the airborne spore concentrations of Ganoderma sp.—one of the most abundant fungal taxa in the area—were developed. Aerobiological sampling was conducted over 2004-2008 using a volumetric Lanzoni trap. Simultaneously, the following meteorological parameters were recorded: daily level of precipitation, maximum and average wind speed, relative humidity and maximum, minimum, average and dew point temperatures. These data were used as the explaining variables. Due to the non-linearity and non-normality of the data set, the applied modelling techniques were artificial neural networks (ANN) and mutlivariate regression trees (MRT). The obtained classification and MRT models predicted threshold conditions above which Ganoderma sp. appeared in the air. It turned out that dew point temperature was the main factor influencing the presence or absence of Ganoderma sp. spores. Further analysis of spore seasons revealed that the airborne fungal spore concentration depended only slightly on meteorological factors.

[Sporogenesis, sporoderm and mature spore ornamentation in Lycopodiaceae].

PubMed

Rincon Baron, Edgar Javier; Rolleri, Cristina Hilda; Passarelli, Lilian M; Espinosa Matías, Silvia; Torres, Alba Marina

2014-09-01

Studies on reproductive aspects, spore morphology and ultrastructure of Lycopodiaceae are not very common in the scientific literature, and constitute essential information to support taxonomic and systematic relationships among the group. In order to complete existing information, adding new and broader contributions on these topics, a comparative analysis of the sporogenesis ultrastructure, with emphasis on cytological aspects of the sporocyte coat development, tapetum, monoplastidic and polyplastidic meiosis, sporoderm ontogeny and ornamentation of the mature spores, was carried out in 43 taxa of eight genera of the Lycopodiaceae: Austrolycopodium, Diphasium, Diphasiastrum, Huperzia (including Phlegmariurus), Lycopodium, Lycopodiella, Palhinhaea and Pseudolycopodiella growing in the Andes of Colombia and the Neotropics. For this study, the transmission elec- tron microscopy (TEM) samples were collected in Cauca and Valle del Cauca Departments, while most of the spores for scanning electron microscopy (SEM) analysis were obtained from herbarium samples. We followed standard preparation procedures for spore observation by TEM and SEM. Results showed that the sporocyte coat is largely composed by primary wall components; the sporocyte develop much of their metabolic activity in the production of their coat, which is retained until the spores release; protective functions for the diploid cells undergoing meiosis is postulated here for this layer. The abundance of dictyosomes in the sporocyte cytoplasm was related to the formation and development of the sporocyte coat. Besides microtubule activity, the membrane of sporocyte folds, associated with electrodense material, and would early determine the final patterns of spore ornamentation. Monoplastidic condition is common in Lycopodium s.l., whereas polyplastidic condition was observed in species of Huperzia and Lycopodiella s. l. In monoplastidic species, the tapetum presents abun- dant multivesicular bodies, while in polyplastidic species, the secretory activity of the tapetum is less intense. Sporoderm development is centripetal, exospore is the first formed layer, then the endospore and, if present, perispore is the final deposited layer. Adult spores of the Lycopodiaceae showed two patterns of ornamentation: negative or caviform (foveolate spores) and positive or muriform ornamentation, the latter with two subtypes (rugate and reticulate spores). The spores of Huperzia are characteristically foveolate, the rugate spores were found in a few species of Huperzia and in all of the Lycopodiella s. l. taxa studied, while Lycopodium s.l. spores bear reticulate ornamentation. Numerous ornamentation traits are diagnostic at the specific level. The types of ornamentation found do not support the recent extreme fragmentation of the family in several genera, but could match, a priori, with the idea of three subfamilies. The findings of sporogenesis, extremely similar in all taxa studied, point more to consider fewer genera, more comprehensive, than the recent, marked splitting of the family.

Production of ochratoxin A by Aspergillus ochraceus NRRL-3174 before and after exposures to 60Co irradiation.

PubMed Central

Applegate, K L; Chipley, J R

1976-01-01

Spores from the toxigenic organism Aspergillus ochraceus NRRL-3174 were exposed to specific levels of gamma irradiation and then allowed to germinate on selected media. Increases in ochratoxin A production by irradiated, compared to non-irradiated, spores were observed after inoculation of spores onto a cracked red wheat or into a synthetic liquid medium. Variations in daily ochratoxin production were also observed for control and irradiated spore-derived cultures developing on both media, with maximum toxin production varying from 7 to 11 days of incubation. The most notable increases in ochratoxin A production occurred from cultures developing from spores having been irradiated with 10, 25, or 50 krad. Exposures to 400 or 600 krad resulted in complete inhibition of spore germination and, consequently, no ochratoxin production. Of the two substrates used, wheat and synthetic, the quantities of ochratoxin A produced were significantly lower in the synthetic media than on the natural substrate. Higher and more rapid toxin production occurred from spores having been irradiated with 10, 25, 50, and 100 krad than occurred from the non-irradiated control spores when grown on synthetic media. Cultures derived from spores having been exposed to 10, 25, 50, and 100 krad produced significantly higher levels of ochratoxin A after 8 days of incubation on natural substrate than did the controls. Analysis of variance revealed that substrate, length of incubation, as well as irradiation levels all affected the time required to produce maximum levels of ochratoxin A. PMID:938031

Antimicrobial effects of gold/copper sulphide (Gold/Copper monosulfide) core/shell nanoparticles on Bacillus anthracis spores and cells

NASA Astrophysics Data System (ADS)

Addae, Ebenezer

Bacillus anthracis is a gram positive, rod shaped and spore forming bacteria. It causes anthrax, a deadly human and animal disease that can kill its victims in three days. The spores of B. anthracis can survive extreme environmental conditions for decades and germinate when exposed to proper conditions. Due to its potential as a bio-weapon, effective disinfectants that pose less harm to the environment and animals are urgently needed. Metal nanoparticles have the potential of killing microbial cells and spores. We present here the effect of Gold/Copper Sulphide core/shell (Au/CuS) nanoparticles on B. anthracis cells and spores. The results indicated that the continuous presence of 0.83 microM during the spore growth in nutrient medium completely inhibited spore outgrowth. Au/CuS nanoparticles at concentration of 4.15 μM completely inactivated B. anthracis cells (x 107) after 30 min of pre-treatment in any of the three buffers including water, PBS, and nutrient broth. However, the same and even higher concentrations of nanoparticles produce no significant spore (x 105) killing after 24 h of pre-treatment. SEM imaging, EDS analysis, and DNA extrusion experiments revealed that nanoparticles damaged the cell membrane causing DNA and cytosolic content efflux and eventually cell death. The study demonstrated the strong antimicrobial activity of Au/CuS nanoparticles to B. anthracis cells and revealed that Au/CuS NPs showed more effective inactivation effect against the cells than they did against the spores.

Effect of sublethal heat treatment on the later stage of germination-to-outgrowth of Clostridium perfringens spores.

PubMed

Sakanoue, Hideyo; Yasugi, Mayo; Miyake, Masami

2018-05-04

Sublethal heating of spores has long been known to stimulate or activate germination, but the underlying mechanisms are not yet fully understood. In this study, we visualized the entire germination-to-outgrowth process of spores from an anaerobic sporeformer, C. perfringens, at single-cell resolution. Quantitative analysis revealed that sublethal heating significantly reduced the time from completion of germination to the beginning of the first cell division. The results indicate that sublethal heating of C. perfringens spores not only sensitizes the responsiveness of germinant receptors but also directly or indirectly facilitates multiple steps during the bacterial regrowth process. © 2018 The Societies and John Wiley & Sons Australia, Ltd.

Biomarkers of Aspergillus spores

NASA Astrophysics Data System (ADS)

Sulc, Miroslav; Peslova, Katerina; Zabka, Martin; Hajduch, Marian; Havlicek, Vladimir

2009-02-01

We applied both matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometric and 1D sodium dodecylsulfate polyacrylamide gel electrophoretic (1D-PAGE) approaches for direct analysis of intact fungal spores of twenty four Aspergillus species. In parallel, we optimized various protocols for protein extraction from Aspergillus spores using acidic conditions, step organic gradient and variable sonication treatment. The MALDI-TOF mass spectra obtained from optimally prepared samples provided a reproducible fingerprint demonstrating the capability of the MALDI-TOF approach to type and characterize different fungal strains within the Aspergillus genus. Mass spectra of intact fungal spores provided signals mostly below 20 kDa. The minimum material amount represented 0.3 [mu]g (10,000 spores). Proteins with higher molecular weight were detected by 1D-PAGEE Eleven proteins were identified from three selected strains in the range 5-25 kDa by the proteomic approach. Hemolysin and hydrophobin have the highest relevance in host-pathogen interactions.

Effect of environmental molds on risk of death from asthma during the pollen season.

PubMed

Targonski, P V; Persky, V W; Ramekrishnan, V

1995-05-01

Many studies have noted an association of ambient aeroallergen levels with exacerbation of asthma. This study was undertaken to examine the relationship of aeroallergen levels with asthma-related mortality in Chicago. The association of environmental aeroallergen levels with death caused by asthma among 5- to 34-year-olds in Chicago was examined for the period of 1985 through 1989. Logistic regression analysis was used to compare the probability of a death caused by asthma occurring on the basis of environmental tree, grass, or ragweed pollen and mold spore levels. Mean mold spore levels but not tree, grass, or ragweed pollen levels were significantly higher for days on which asthma-related death occurred than for days on which no deaths occurred (z = 2.80, p < 0.005). The odds of a death caused by asthma occurring on days with mold spore counts of 1000 spores per cubic meter or greater was 2.16 times higher (95% confidence interval = 1.31, 3.56, p = 0.003) than on days on which mold spore counts were less than 1000 spores per cubic meter. The association with mold spore levels remained significant on multivariate logistic regression with mold spore counts measured as a continuous variable and controlling for pollens, with the odds of an asthma-related death occurring being 1.2 times higher (95% confidence interval = 1.07-1.34) for every increase of 1000 spores per cubic meter in daily mold spore levels. Although death caused by asthma also involves personal, social, and medical access factors, these data suggest that exposure to environmental molds may play a role in asthma-related mortality and should be considered in prevention strategies.

Impact of spores on the comparative efficacies of five antibiotics for treatment of Bacillus anthracis in an in vitro hollow fiber pharmacodynamic model.

PubMed

Louie, Arnold; VanScoy, Brian D; Brown, David L; Kulawy, Robert W; Heine, Henry S; Drusano, George L

2012-03-01

Bacillus anthracis, the bacterium that causes anthrax, is an agent of bioterrorism. The most effective antimicrobial therapy for B. anthracis infections is unknown. An in vitro pharmacodynamic model of B. anthracis was used to compare the efficacies of simulated clinically prescribed regimens of moxifloxacin, linezolid, and meropenem with the "gold standards," doxycycline and ciprofloxacin. Treatment outcomes for isogenic spore-forming and non-spore-forming strains of B. anthracis were compared. Against spore-forming B. anthracis, ciprofloxacin, moxifloxacin, linezolid, and meropenem reduced the B. anthracis population by 4 log(10) CFU/ml over 10 days. Doxycycline reduced the population of this B. anthracis strain by 5 log(10) CFU/ml (analysis of variance [ANOVA] P = 0.01 versus other drugs). Against an isogenic non-spore-forming strain, meropenem killed the vegetative B. anthracis the fastest, followed by moxifloxacin and ciprofloxacin and then doxycycline. Linezolid offered the lowest bacterial kill rate. Heat shock studies using the spore-producing B. anthracis strain showed that with moxifloxacin, ciprofloxacin, and meropenem therapies the total population was mostly spores, while the population was primarily vegetative bacteria with linezolid and doxycycline therapies. Spores have a profound impact on the rate and extent of killing of B. anthracis. Against spore-forming B. anthracis, the five antibiotics killed the total (spore and vegetative) bacterial population at similar rates (within 1 log(10) CFU/ml of each other). However, bactericidal antibiotics killed vegetative B. anthracis faster than bacteriostatic drugs. Since only vegetative-phase B. anthracis produces the toxins that may kill the infected host, the rate and mechanism of killing of an antibiotic may determine its overall in vivo efficacy. Further studies are needed to examine this important observation.

Impact of Spores on the Comparative Efficacies of Five Antibiotics for Treatment of Bacillus anthracis in an In Vitro Hollow Fiber Pharmacodynamic Model

PubMed Central

VanScoy, Brian D.; Brown, David L.; Kulawy, Robert W.; Heine, Henry S.; Drusano, George L.

2012-01-01

Bacillus anthracis, the bacterium that causes anthrax, is an agent of bioterrorism. The most effective antimicrobial therapy for B. anthracis infections is unknown. An in vitro pharmacodynamic model of B. anthracis was used to compare the efficacies of simulated clinically prescribed regimens of moxifloxacin, linezolid, and meropenem with the “gold standards,” doxycycline and ciprofloxacin. Treatment outcomes for isogenic spore-forming and non-spore-forming strains of B. anthracis were compared. Against spore-forming B. anthracis, ciprofloxacin, moxifloxacin, linezolid, and meropenem reduced the B. anthracis population by 4 log10 CFU/ml over 10 days. Doxycycline reduced the population of this B. anthracis strain by 5 log10 CFU/ml (analysis of variance [ANOVA] P = 0.01 versus other drugs). Against an isogenic non-spore-forming strain, meropenem killed the vegetative B. anthracis the fastest, followed by moxifloxacin and ciprofloxacin and then doxycycline. Linezolid offered the lowest bacterial kill rate. Heat shock studies using the spore-producing B. anthracis strain showed that with moxifloxacin, ciprofloxacin, and meropenem therapies the total population was mostly spores, while the population was primarily vegetative bacteria with linezolid and doxycycline therapies. Spores have a profound impact on the rate and extent of killing of B. anthracis. Against spore-forming B. anthracis, the five antibiotics killed the total (spore and vegetative) bacterial population at similar rates (within 1 log10 CFU/ml of each other). However, bactericidal antibiotics killed vegetative B. anthracis faster than bacteriostatic drugs. Since only vegetative-phase B. anthracis produces the toxins that may kill the infected host, the rate and mechanism of killing of an antibiotic may determine its overall in vivo efficacy. Further studies are needed to examine this important observation. PMID:22155821

Comparison of innovative molecular approaches and standard spore assays for assessment of surface cleanliness.

PubMed

Cooper, Moogega; La Duc, Myron T; Probst, Alexander; Vaishampayan, Parag; Stam, Christina; Benardini, James N; Piceno, Yvette M; Andersen, Gary L; Venkateswaran, Kasthuri

2011-08-01

A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces.

Molecular Markers to Detect the Formation of Heterokaryon and Homokaryon from Asexual Spores of the Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes).

PubMed

Wang, Hong; Cai, Tao; Wei, Jing; Feng, Aiping; Lin, Nan; Bao, Dapeng

2015-01-01

Cordyceps militaris is widely cultivated on artificial media in China; however, the cultures often are afflicted with the degeneration of nonfruiting strains. To understand the mechanism of degeneration of C. militaris, from the heterokaryotic strain into the homokaryotic strain, we examined the mating-type genes present in individual asexual spores. Further, we determined the distribution ratio of the different mating-type genes among a sample of asexual spores and the growth rate of heterokaryotic and homokaryotic strains of C. militaris. The distribution ratio of 3 groups of asexual spores from C. militaris heterokaryotic strains was determined as 1:1:1 by statistical analysis, whereas that of the two types of nuclei among asexual spores was 1:1. Nearly two-thirds of the asexual spore isolates were homokaryon, which showed a growth speed similar to the heterokaryon. However, the homokaryon (bearing mating-type MAT-HMG) grew significantly faster at times compared with the heterokaryon. Therefore, the purity of the spawn was difficult to establish. C. militaris heterokaryotic strains can transform into a homokaryotic strain following continued subculture.

Label-free Chemical Imaging of Fungal Spore Walls by Raman Microscopy and Multivariate Curve Resolution Analysis

PubMed Central

Noothalapati, Hemanth; Sasaki, Takahiro; Kaino, Tomohiro; Kawamukai, Makoto; Ando, Masahiro; Hamaguchi, Hiro-o; Yamamoto, Tatsuyuki

2016-01-01

Fungal cell walls are medically important since they represent a drug target site for antifungal medication. So far there is no method to directly visualize structurally similar cell wall components such as α-glucan, β-glucan and mannan with high specificity, especially in a label-free manner. In this study, we have developed a Raman spectroscopy based molecular imaging method and combined multivariate curve resolution analysis to enable detection and visualization of multiple polysaccharide components simultaneously at the single cell level. Our results show that vegetative cell and ascus walls are made up of both α- and β-glucans while spore wall is exclusively made of α-glucan. Co-localization studies reveal the absence of mannans in ascus wall but are distributed primarily in spores. Such detailed picture is believed to further enhance our understanding of the dynamic spore wall architecture, eventually leading to advancements in drug discovery and development in the near future. PMID:27278218

Use of aerobic spores as a surrogate for cryptosporidium oocysts in drinking water supplies.

PubMed

Headd, Brendan; Bradford, Scott A

2016-03-01

Waterborne illnesses are a growing concern among health and regulatory agencies worldwide. The United States Environmental Protection Agency has established several rules to combat the contamination of water supplies by cryptosporidium oocysts, however, the detection and study of cryptosporidium oocysts is hampered by methodological and financial constraints. As a result, numerous surrogates for cryptosporidium oocysts have been proposed by the scientific community and efforts are underway to evaluate many of the proposed surrogates. The purpose of this review is to evaluate the suitability of aerobic bacterial spores to serve as a surrogate for cryptosporidium oocysts in identifying contaminated drinking waters. To accomplish this we present a comparison of the biology and life cycles of aerobic spores and oocysts and compare their physical properties. An analysis of their surface properties is presented along with a review of the literature in regards to the transport, survival, and prevalence of aerobic spores and oocysts in the saturated subsurface environment. Aerobic spores and oocysts share many commonalities with regard to biology and survivability, and the environmental prevalence and ease of detection make aerobic spores a promising surrogate for cryptosporidium oocysts in surface and groundwater. However, the long-term transport and release of aerobic spores still needs to be further studied, and compared with available oocyst information. In addition, the surface properties and environmental interactions of spores are known to be highly dependent on the spore taxa and purification procedures, and additional research is needed to address these issues in the context of transport. Published by Elsevier Ltd.

Prevalence of culturable airborne spores of selected allergenic and pathogenic fungi in outdoor air

NASA Astrophysics Data System (ADS)

O'Gorman, Céline M.; Fuller, Hubert T.

2008-06-01

Temporal and spatial variations in airborne spore concentrations of selected allergenic and pathogenic fungi were examined in Dublin, Ireland, in 2005. Air samples were taken at four outdoor locations in the city every 2 weeks, coupled with measurements of meteorological conditions. Total culturable airborne fungal spore concentrations in Dublin ranged from 30-6800 colony forming units per cubic metre of air (CFU m-3) over the 12-month period. Cladosporium, Penicillium, Aspergillus and Alternaria spores were constantly present in the Dublin atmosphere, representing >20% of the total culturable spore count. Concentrations of Cladosporium increased significantly in summer and reached allergenic threshold levels, peaking at over 3200 CFU m-3 in August. Penicillium spore concentrations never reached allergenic threshold levels, with average concentrations of <150 CFU m-3. Alternaria conidia formed only 0.3% of the total culturable fungal spore count and concentrations never exceeded 50 CFU m-3, attributable to the coastal position of Dublin and its low levels of arable production. The opportunistic human pathogen Aspergillus fumigatus was present throughout the year in nominal concentrations (<10 CFU m-3), but sporadic high counts were also recorded (300-400 CFU m-3), the potential health implications of which give cause for concern. Spores of neither Cryptococcus neoformans nor Stachybotrys chartarum were detected, but airborne basidiospores of Schizophyllum commune were evidenced by the dikaryotization of monokaryon tester strains following exposure to the air. The relationships between airborne fungal spore concentrations and meteorological factors were analysed by redundancy analysis and revealed positive correlations between temperature and Cladosporium and relative humidity and Penicillium and Aspergillus.

Glycoconjugates Effects: do Gender and Ethnicity Influence Exposure of Pathogen by Peripheral Mononuclear Cells ?

NASA Astrophysics Data System (ADS)

Lahiani, Mohamed; Tarasenko, Olga

2010-04-01

Members of the Bacillus cereus group demonstrate different pathological effects. B. cereus is a spore-forming, gram positive bacterium responsible for most foodborne illnesses. It was shown that susceptibility to infection and response to vaccines or treatments can be attributed to specific immunogenetic factors including gender and ethnicity. Glycoconjugate polymers (GCs) are potentially important in pharmaceutical and biomedical research. Our group has shown that GCs activate murine macrophages and promote killing of Bacillus cereus spores during phagocytosis. We hypothesized that the GCs effects are independent from gender and race. The goal of the present study was two-folds: A) determine whether GCs influence on human PMNC exposure of B. cereus spores and B) analyze whether gender and ethnicity influence of the effect of GCs. GCs were studied during exposure and post-exposure conditions. Phagocytosis was performed during exposure of PMNC to Bacillus spores. Post-exposure analysis involved cytotoxicity, cell viability and activation, and colonies forming unit. GC1 and GC3 enhance Bacillus spore killing. GC1 proved more effective than GC3 in spore killing while activating PMNC. Results demonstrate GCs effect were independent from ethnicity or gender. Findings of this research demonstrated that GC can be used as ligands to stimulate PMNC and kill B. cereus spores.

Conserved oligopeptide permeases modulate sporulation initiation in Clostridium difficile.

PubMed

Edwards, Adrianne N; Nawrocki, Kathryn L; McBride, Shonna M

2014-10-01

The anaerobic gastrointestinal pathogen Clostridium difficile must form a metabolically dormant spore to survive in oxygenic environments and be transmitted from host to host. The regulatory factors by which C. difficile initiates and controls the early stages of sporulation in C. difficile are not highly conserved in other Clostridium or Bacillus species. Here, we investigated the role of two conserved oligopeptide permeases, Opp and App, in the regulation of sporulation in C. difficile. These permeases are known to positively affect sporulation in Bacillus species through the import of sporulation-specific quorum-sensing peptides. In contrast to other spore-forming bacteria, we discovered that inactivating these permeases in C. difficile resulted in the earlier expression of early sporulation genes and increased sporulation in vitro. Furthermore, disruption of opp and app resulted in greater virulence and increased the amounts of spores recovered from feces in the hamster model of C. difficile infection. Our data suggest that Opp and App indirectly inhibit sporulation, likely through the activities of the transcriptional regulator SinR and its inhibitor, SinI. Taken together, these results indicate that the Opp and App transporters serve a different function in controlling sporulation and virulence in C. difficile than in Bacillus subtilis and suggest that nutrient availability plays a significant role in pathogenesis and sporulation in vivo. This study suggests a link between the nutritional status of the environment and sporulation initiation in C. difficile. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A public health issue related to collateral seismic hazards: The valley fever outbreak triggered by the 1994 Northridge, California earthquake

USGS Publications Warehouse

Jibson, R.W.

2002-01-01

Following the 17 January 1994 Northridge. California earthquake (M = 6.7), Ventura County, California, experienced a major outbreak of coccidioidomycosis (CM), commonly known as valley fever, a respiratory disease contracted by inhaling airborne fungal spores. In the 8 weeks following the earthquake (24 January through 15 March), 203 outbreak-associated cases were reported, which is about an order of magnitude more than the expected number of cases, and three of these cases were fatal. Simi Valley, in easternmost Ventura County, had the highest attack rate in the county, and the attack rate decreased westward across the county. The temporal and spatial distribution of CM cases indicates that the outbreak resulted from inhalation of spore-contaminated dust generated by earthquake-triggered landslides. Canyons North East of Simi Valley produced many highly disrupted, dust-generating landslides during the earthquake and its aftershocks. Winds after the earthquake were from the North East, which transported dust into Simi Valley and beyond to communities to the West. The three fatalities from the CM epidemic accounted for 4 percent of the total earthquake-related fatalities.

Antifungal activity of colistin against mucorales species in vitro and in a murine model of Rhizopus oryzae pulmonary infection.

PubMed

Ben-Ami, Ronen; Lewis, Russell E; Tarrand, Jeffrey; Leventakos, Konstantinos; Kontoyiannis, Dimitrios P

2010-01-01

In immunosuppressed hosts, mucormycosis is a life-threatening infection with few treatment options. We studied the activity of colistin (polymyxin E) against Mucorales species in vitro and in a murine model of pulmonary Rhizopus oryzae infection. Colistin exhibited fungicidal activity in vitro against Mucorales spores and mycelia. At the colistin MIC, initial R. oryzae hyphal damage was followed by rapid regrowth; however, regrowth was prevented by combining colistin with a subinhibitory concentration of amphotericin B. Using electron microscopy and FM4-64 staining, we demonstrated that colistin disrupts R. oryzae cytoplasmic and vacuolar membranes, resulting in the leakage of intracellular contents. The prophylactic intranasal treatment of immunosuppressed mice with colistimethate significantly reduced the mortality rate and pulmonary fungal burden resulting from inhalational challenge with R. oryzae spores, whereas intraperitoneal colistimethate treatment had no effect. We conclude that colistin has modest in vitro and in vivo fungicidal activity against Mucorales spp. Further studies are warranted to assess the use of this drug in the prevention and treatment of mucormycosis.

Antifungal Activity of Colistin against Mucorales Species In Vitro and in a Murine Model of Rhizopus oryzae Pulmonary Infection▿

PubMed Central

Ben-Ami, Ronen; Lewis, Russell E.; Tarrand, Jeffrey; Leventakos, Konstantinos; Kontoyiannis, Dimitrios P.

2010-01-01

In immunosuppressed hosts, mucormycosis is a life-threatening infection with few treatment options. We studied the activity of colistin (polymyxin E) against Mucorales species in vitro and in a murine model of pulmonary Rhizopus oryzae infection. Colistin exhibited fungicidal activity in vitro against Mucorales spores and mycelia. At the colistin MIC, initial R. oryzae hyphal damage was followed by rapid regrowth; however, regrowth was prevented by combining colistin with a subinhibitory concentration of amphotericin B. Using electron microscopy and FM4-64 staining, we demonstrated that colistin disrupts R. oryzae cytoplasmic and vacuolar membranes, resulting in the leakage of intracellular contents. The prophylactic intranasal treatment of immunosuppressed mice with colistimethate significantly reduced the mortality rate and pulmonary fungal burden resulting from inhalational challenge with R. oryzae spores, whereas intraperitoneal colistimethate treatment had no effect. We conclude that colistin has modest in vitro and in vivo fungicidal activity against Mucorales spp. Further studies are warranted to assess the use of this drug in the prevention and treatment of mucormycosis. PMID:19858263

A Public Health Issue Related To Collateral Seismic Hazards: The Valley Fever Outbreak Triggered By The 1994 Northridge, California Earthquake

NASA Astrophysics Data System (ADS)

Jibson, Randall W.

Following the 17 January 1994 Northridge, California earthquake (M = 6.7), Ventura County, California, experienced a major outbreak ofcoccidioidomycosis (CM), commonly known as valley fever, a respiratory disease contracted byinhaling airborne fungal spores. In the 8 weeks following the earthquake (24 Januarythrough 15 March), 203 outbreak-associated cases were reported, which is about an order of magnitude more than the expected number of cases, and three of these cases were fatal.Simi Valley, in easternmost Ventura County, had the highest attack rate in the county,and the attack rate decreased westward across the county. The temporal and spatial distribution of CM cases indicates that the outbreak resulted from inhalation of spore-contaminated dust generated by earthquake-triggered landslides. Canyons North East of Simi Valleyproduced many highly disrupted, dust-generating landslides during the earthquake andits aftershocks. Winds after the earthquake were from the North East, which transporteddust into Simi Valley and beyond to communities to the West. The three fatalities from the CM epidemic accounted for 4 percent of the total earthquake-related fatalities.

Genetic analysis reveals efficient sexual spore dispersal at a fine spatial scale in Armillaria ostoyae, the causal agent of root-rot disease in conifers.

PubMed

Dutech, Cyril; Labbé, Frédéric; Capdevielle, Xavier; Lung-Escarmant, Brigitte

Armillaria ostoyae (sometimes named Armillaria solidipes) is a fungal species causing root diseases in numerous coniferous forests of the northern hemisphere. The importance of sexual spores for the establishment of new disease centres remains unclear, particularly in the large maritime pine plantations of southwestern France. An analysis of the genetic diversity of a local fungal population distributed over 500 ha in this French forest showed genetic recombination between genotypes to be frequent, consistent with regular sexual reproduction within the population. The estimated spatial genetic structure displayed a significant pattern of isolation by distance, consistent with the dispersal of sexual spores mostly at the spatial scale studied. Using these genetic data, we inferred an effective density of reproductive individuals of 0.1-0.3 individuals/ha, and a second moment of parent-progeny dispersal distance of 130-800 m, compatible with the main models of fungal spore dispersal. These results contrast with those obtained for studies of A. ostoyae over larger spatial scales, suggesting that inferences about mean spore dispersal may be best performed at fine spatial scales (i.e. a few kilometres) for most fungal species. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Genome size evolution in Ontario ferns (Polypodiidae): evolutionary correlations with cell size, spore size, and habitat type and an absence of genome downsizing.

PubMed

Henry, Thomas A; Bainard, Jillian D; Newmaster, Steven G

2014-10-01

Genome size is known to correlate with a number of traits in angiosperms, but less is known about the phenotypic correlates of genome size in ferns. We explored genome size variation in relation to a suite of morphological and ecological traits in ferns. Thirty-six fern taxa were collected from wild populations in Ontario, Canada. 2C DNA content was measured using flow cytometry. We tested for genome downsizing following polyploidy using a phylogenetic comparative analysis to explore the correlation between 1Cx DNA content and ploidy. There was no compelling evidence for the occurrence of widespread genome downsizing during the evolution of Ontario ferns. The relationship between genome size and 11 morphological and ecological traits was explored using a phylogenetic principal component regression analysis. Genome size was found to be significantly associated with cell size, spore size, spore type, and habitat type. These results are timely as past and recent studies have found conflicting support for the association between ploidy/genome size and spore size in fern polyploid complexes; this study represents the first comparative analysis of the trend across a broad taxonomic group of ferns.

Biomimetic TiO2 formation from interfacial sol-gel chemistry leading to new photocatalysts

NASA Astrophysics Data System (ADS)

Jaffer Al-Timimi, Iman A.; Onwukwe, Uche K.; Worsley, Myles P.; Sermon, Paul A.

2016-09-01

The surfaces of Portobello mushroom spores (PMS) have been used to produce Au and Ag nanoparticles, which are held thereon. They have then been overcoated with TiOx. These adsorbed more methyl orange (MO) pollutant from water than commercial P25 TiO2. After calcination they form biomimetic TiO2 (PMS) and removal of the biotemplate, they catalyse faster rates of MO from water (molecules/mg/s) than P25 anataserutile. Other biotemplates are now anticipated that will yield biomimetic photocatalysts with higher turnover number (s-1) removal of endocrine disrupters from water.

Dmc1 of Schizosaccharomyces pombe plays a role in meiotic recombination.

PubMed

Fukushima, K; Tanaka, Y; Nabeshima, K; Yoneki, T; Tougan, T; Tanaka, S; Nojima, H

2000-07-15

We report here a Schizosaccharomyces pombe gene (dmc1(+)) that resembles budding yeast DMC1 in the region immediately upstream of the rad24(+) gene. We showed by northern and Southern blot analysis that dmc1(+) and rad24(+) are co-transcribed as a bicistronic mRNA of 2.8 kb with meiotic specificity, whereas rad24(+) itself is constitutively transcribed as a 1.0-kb mRNA species during meiosis. Induction of the bicistronic transcript is under the control of a meiosis-specific transcription factor, Ste11. Disruption of both dmc1(+) and rad24(+) had no effect on mitosis or spore formation, and dmc1Delta cells displayed no change in sensitivity to UV or gamma irradiation relative to the wild type. Tetrad analysis indicated that Dmc1 is involved in meiotic recombination. Analysis of gene conversion frequencies using single and double mutants of dmc1 and rhp51 indicated that both Dmc1 and Rhp51 function in meiotic gene conversion. These observations, together with a high level of sequence identity, indicate that the dmc1(+) gene of S. POMBE: is a structural homolog of budding yeast DMC1, sharing both similar and distinct functions in meiosis.

Mechanism of the hydrolysis of 4-methylumbelliferyl-beta-D-glucoside by germinating and outgrowing spores of Bacillus species.

PubMed

Setlow, B; Cabrera-Martinez, R-M; Setlow, P

2004-01-01

To determine the mechanism of the hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside (beta-MUG) by germinating and outgrowing spores of Bacillus species. Spores of B. atrophaeus (formerly B. subtilis var. niger, Fritze and Pukall 2001) are used as biological indicators of the efficacy of ethylene oxide sterilization by measurement of beta-MUG hydrolysis during spore germination and outgrowth. It was previously shown that beta-MUG is hydrolysed to 4-methylumbelliferone (MU) during the germination and outgrowth of B. atrophaeus spores (Chandrapati and Woodson 2003), and this was also the case with spores of B. subtilis 168. Germination of spores of either B. atrophaeus or B. subtilis with chloramphenicol reduced beta-MUG hydrolysis by almost 99%, indicating that proteins needed for rapid beta-MUG hydrolysis are synthesized during spore outgrowth. However, the residual beta-MUG hydrolysis during spore germination with chloramphenicol indicated that dormant spores contain low levels of proteins needed for beta-MUG uptake and hydrolysis. With B. subtilis 168 spores that lacked several general proteins of the phosphotransferase system (PTS) for sugar uptake, beta-MUG hydrolysis during spore germination and outgrowth was decreased >99.9%. This indicated that beta-MUG is taken up by the PTS, resulting in the intracellular accumulation of the phosphorylated form of beta-MUG, beta-MUG-6-phosphate (beta-MUG-P). This was further demonstrated by the lack of detectable glucosidase activity on beta-MUG in dormant, germinated and outgrowing spore extracts, while phosphoglucosidase active on beta-MUG-P was readily detected. Dormant B. subtilis 168 spores had low levels of at least four phosphoglucosidases active on beta-MUG-P: BglA, BglH, BglC (originally called YckE) and BglD (originally called YdhP). These enzymes were also detected in spores germinating and outgrowing with beta-MUG, but levels of BglH were the highest, as this enzyme's synthesis was induced ca 100-fold during spore outgrowth in the presence of beta-MUG. Deletion of the genes coding for BglA, BglH, BglC and BglD reduced beta-MUG hydrolysis by germinating and outgrowing spores of B. subtilis 168 at least 99.7%. Assay of glucosidases active on beta-MUG or beta-MUG-P in extracts of dormant and outgrowing spores of B. atrophaeus revealed no enzyme active on beta-MUG and one enzyme that comprised > or =90% of the phosphoglucosidase active on beta-MUG-P. Partial purification and amino-terminal sequence analysis of this phosphoglucosidase identified this enzyme as BglH. Generation of MU from beta-MUG by germinating and outgrowing spores of B. atrophaeus and B. subtilis is mediated by the PTS-driven uptake and phosphorylation of beta-MUG, followed by phosphoglucosidase action on the intracellular beta-MUG-P. The major phosphoglucosidase catalyzing MU generation from beta-MUG-P in spores of both species is probably BglH. This work provides new insight into the mechanism of uptake and hydrolysis of beta-MUG by germinating and outgrowing spores of Bacillus species, in particular B. atrophaeus. The research reported here provides a biological basis for a Rapid Readout Biological Indicator that is used to monitor the efficacy of ethylene oxide sterilization.

Studies on a Factor in Sweet Potato Root Which Agglutinates Spores of Ceratocystis fimbriata, Black Rot Fungus 1

PubMed Central

Kojime, Mineo; Kawakita, Kazuhito; Uritani, Ikuzo

1982-01-01

A factor which agglutinated the spores of Ceratocystis fimbriata in the presence of Ca2+ was purified from sweet potato (Ipomea batatas Lam cv. Norin[1]) root. Element composition of the purified factor was as follows; analysis found: C (29.8%), H (3.97%), O (65.34%), N (0.81%): calculated for C43H69O70N1: C (30.02%), H (4.01%), O (65.15%), N (0.81%). The factor was mainly composed of galacturonic acid (53% of dry weight) and contained arabinose, fucose, and unidentified component as minor components. The factor also agglutinated A-, B-, AB-, and O types of human erythrocytes to almost the same degree in the presence of Ca2+. The differential spore-agglutinating activity of the factor depended on the pH of the assay medium; it agglutinated similarly the germinated spores of sweet potato and coffee strains at pH 7.5 and 5.5, whereas it displayed a distinct differential agglutinating activity at pH 6.5. The factor was assayed for spore-agglutinating activity at pH 6.5, using the germinated and ungerminated spores of seven strains of C. fimbriata; sweet potato, coffee, prune, cacao, oak, taro, and almond strains. The factor agglutinated ungerminated spores of all seven strains similarly, although small differences were observed among strains. On the other hand, a clear differential agglutination was observed among the germinated spores of various strains; sweet potato and almond strains were highly insensitive in comparison with other strains. The growth of the agglutinated spores of C. fimbriata was inhibited. These results are discussed in relation to host-parasite specificity. Images PMID:16662232

Comparison of fungal spores concentrations measured with wideband integrated bioaerosol sensor and Hirst methodology

NASA Astrophysics Data System (ADS)

Fernández-Rodríguez, S.; Tormo-Molina, R.; Lemonis, N.; Clot, B.; O'Connor, D. J.; Sodeau, John R.

2018-02-01

The aim of this work was to provide both a comparison of traditional and novel methodologies for airborne spores detection (i.e. the Hirst Burkard trap and WIBS-4) and the first quantitative study of airborne fungal concentrations in Payerne (Western Switzerland) as well as their relation to meteorological parameters. From the traditional method -Hirst trap and microscope analysis-, sixty-three propagule types (spores, sporangia and hyphae) were identified and the average spore concentrations measured over the full period amounted to 4145 ± 263.0 spores/m3. Maximum values were reached on July 19th and on August 6th. Twenty-six spore types reached average levels above 10 spores/m3. Airborne fungal propagules in Payerne showed a clear seasonal pattern, increasing from low values in early spring to maxima in summer. Daily average concentrations above 5000 spores/m3 were almost constant in summer from mid-June onwards. Weather parameters showed a relevant role for determining the observed spore concentrations. Coniferous forest, dominant in the surroundings, may be a relevant source for airborne fungal propagules as their distribution and predominant wind directions are consistent with the origin. The comparison between the two methodologies used in this campaign showed remarkably consistent patterns throughout the campaign. A correlation coefficient of 0.9 (CI 0.76-0.96) was seen between the two over the time period for daily resolutions (Hirst trap and WIBS-4). This apparent co-linearity was seen to fall away once increased resolution was employed. However at higher resolutions upon removal of Cladosporium species from the total fungal concentrations (Hirst trap), an increased correlation coefficient was again noted between the two instruments (R = 0.81 with confidence intervals of 0.74 and 0.86).

Increased levels of ambient fungal spores in Taiwan are associated with dust events from China

NASA Astrophysics Data System (ADS)

Wu, Pei-Chih; Tsai, Jui-Chen; Li, Fang-Chun; Lung, Shih-Chun; Su, Huey-Jen

2004-09-01

Fungi are ubiquitous in nature and their spores are often dispersed into the atmosphere through turbulent airstreams. As yellow sandstorm blown from deserts in China had affected the ambient air quality with increasing levels of ambient particulates, often including significant amounts of biologically active particles has therefore become imperative for concerns of their health implications. Our study was aimed to examine the effects of yellow sandstorm events on the fungal composition and concentrations in ambient air. Atmospheric fungal spores were continuously collected using Burkard Volumetric Spore Trap. Samples collected between December 2000 and April 2001 were selected for priority analysis from days when the yellow sandstorms were reported to affect Taiwan according to the Central Weather Bureau in Taiwan. The composition of dominant spores such as Basidiospore, Penicillium/Aspergillus, Nigrospora, Arthrinium, Curvularia, Rusts, Stemphylium, Cercospora, Pithomyces, and unidentified fungi were significantly higher than those of background days. The increase of Basidiospore, Penicillium/Aspergillus, Nigrospora, and those unidentified fungi seems to be significantly associated with the increase of ambient particulate levels with regression coefficients ranging from 0.887 to 31.98. Our study has identified increasing ambient concentrations during sandstorm episodes are observed for some major fungi, Basidiospore, Penicillium, Aspergillus, and those unidentified fungi and the trends of the increase seems to associate with ambient particulate levels. Further efforts to clarify the relationship between those high fungal spore exposures and clinical adverse health effects are suggested in the future. In addition, effects of climatic factors and other particulate levels on the variation of ambient fungal spore levels are also desired in further study. Additional monitoring of ambient fungal spores in the first line of west coastline is hoped to assist in differentiating changes of local spores and contribution for sandstorms during the episodes.

Detection of Biomass in New York City Aerosols: Light Scattering and Optical Fluorescence Techniques

NASA Astrophysics Data System (ADS)

Niebauer, M.; Alimova, A.; Katz, A.; Xu, M.; Rudolph, E.; Steiner, J.; Alfano, R. R.

2005-12-01

Optical spectroscopy is an ideal method for detecting bacteria and spores in real time. Optical fluorescence spectroscopy examination of New York City aerosols is used to quantify the mass of bacteria spores present in air masses collected at 14 liters/minute onto silica fiber filters, and on silica fiber ribbons using an Environmental Beta Attenuation Monitor manufactured by MetOne Instruments configured for the PM2.5 fraction. Dipicolinic acid (DPA), a molecule found primarily in bacterial spores, is the most characteristic component of spores in trial experiments on over 200 collected aerosol samples. DPA is extracted from the spores using a heat bath and chelated with Terbium. The DPA:Tb is detected by measuring its characteristic fluorescence with emission bands at 490, 545 and 585 nm for 270 nm excitation. Light scattering also measures the size distribution for a number of a variety of bacteria - Bacillus subtilis (rod shaped), Staphylococcus aureus (spherical) and Pseudomonas aeruginosa (short rods) establishing that optical techniques satisfactorily distinguish populations based on their variable morphology. Size and morphology are obtained by applying a variation of the Gaussian Ray Approximation theory of anomalous diffraction theory to an analysis of the transmission spectra in the range of 0.4 to 1.0 microns. In test experiments, the refractive index of the inner spore core of Bacillus subtilis decreases from 1.51 to 1.39 while the spore radius enlarges from 0.38 to 0.6 micrometers. Optical determinations are verified by oil-immersion techniques and by scanning electron microscope measurements. Characterization of spores, germinating spore materials, and bacteria is considered vital to tracing bacteria in the environment, for the development of life-detection systems for planetary exploration, monitoring pathogens in environmental systems, and for the preparation of anti-terrorism strategies.

The nature of water within bacterial spores: protecting life in extreme environments

NASA Astrophysics Data System (ADS)

Rice, Charles V.; Friedline, Anthony; Johnson, Karen; Zachariah, Malcolm M.; Thomas, Kieth J., III

2011-10-01

The bacterial spore is a formidable container of life, protecting the vital contents from chemical attack, antimicrobial agents, heat damage, UV light degradation, and water dehydration. The exact role of the spore components remains in dispute. Nevertheless, water molecules are important in each of these processes. The physical state of water within the bacterial spore has been investigated since the early 1930's. The water is found two states, free or bound, in two different areas, core and non-core. It is established that free water is accessible to diffuse and exchange with deuterated water and that the diffusible water can access all areas of the spore. The presence of bound water has come under recent scrutiny and has been suggested the water within the core is mobile, rather than bound, based on the analysis of deuterium relaxation rates. Using an alternate method, deuterium quadrupole-echo spectroscopy, we are able to distinguish between mobile and immobile water molecules. In the absence of rapid motion, the deuterium spectrum of D2O is dominated by a broad line, whose line shape is used as a characteristic descriptor of molecular motion. The deuterium spectrum of bacterial spores reveals three distinct features: the broad peak of immobilized water, a narrow line of water in rapid motion, and a signal of intermediate width. This third signal is assigned this peak from partially deuterated proteins with the spore in which N-H groups have undergone exchange with water deuterons to form N-D species. As a result of these observations, the nature of water within the spore requires additional explanation to understand how the spore and its water preserve life.

Fern Spore Longevity in Saline Water: Can Sea Bottom Sediments Maintain a Viable Spore Bank?

PubMed Central

de Groot, G. Arjen; During, Heinjo

2013-01-01

Freshwater and marine sediments often harbor reservoirs of plant diaspores, from which germination and establishment may occur whenever the sediment falls dry. Therewith, they form valuable records of historical inter- and intraspecific diversity, and are increasingly exploited to facilitate diversity establishment in new or restored nature areas. Yet, while ferns may constitute a considerable part of a vegetation’s diversity and sediments are known to contain fern spores, little is known about their longevity, which may suffer from inundation and - in sea bottoms - salt stress. We tested the potential of ferns to establish from a sea or lake bottom, using experimental studies on spore survival and gametophyte formation, as well as a spore bank analysis on sediments from a former Dutch inland sea. Our experimental results revealed clear differences among species. For Asplenium scolopendrium and Gymnocarpium dryopteris, spore germination was not affected by inundated storage alone, but decreased with rising salt concentrations. In contrast, for Asplenium trichomanes subsp. quadrivalens germination decreased following inundation, but not in response to salt. Germination rates decreased with time of storage in saline water. Smaller and less viable gametophytes were produced when saline storage lasted for a year. Effects on germination and gametophyte development clearly differed among genotypes of A. scolopendrium. Spore bank analyses detected no viable spores in marine sediment layers. Only two very small gametophytes (identified as Thelypteris palustris via DNA barcoding) emerged from freshwater sediments. Both died before maturation. We conclude that marine, and likely even freshwater sediments, will generally be of little value for long-term storage of fern diversity. The development of any fern vegetation on a former sea floor will depend heavily on the deposition of spores onto the drained land by natural or artificial means of dispersal. PMID:24223951

Isolation of the Paenibacillus phoenicis, a Spore-Forming Bacterium

NASA Technical Reports Server (NTRS)

Benardini, James N.; Vaishampayan, Parag A.; Venkateswaran, Kasthuri J.; Osman, Shariff; Satomi, Masataka

2010-01-01

A microorganism was isolated from the surfaces of the cleanroom facility in which the Phoenix lander was assembled. The isolated bacterial strain was subjected to a comprehensive polyphasic analysis to characterize its taxonomic position. Both phenotypic and phylogenetic analyses clearly indicate that this isolate belongs to the genus Paenibacillus and represents a novel species. Bacillus spores have been utilized to assess the degree and level of microbiological contamination on spacecraft and their associated spacecraft assembly facilities. Spores of Bacillus species are of particular concern to planetary protection due to the extreme resistance of some members of the genus to space environmental conditions such as UV and gamma radiation, vacuum, oxidation, and temperature fluctuation. These resistive spore phenotypes have enhanced potential for transfer, and subsequent proliferation, of terrestrial microbes on another solar body. Due to decreased nutrient conditions within spacecraft assembly facility clean rooms, the vegetative cells of Bacillus species and other spore-forming Paenibacillus species are induced to sporulate, thereby enhancing their survivability of bioreduction

Analysis of Isoaccepting Transfer Ribonucleic Acid Species of Bacillus subtilis: Chromatographic Differences Between Transfer Ribonucleic Acids from Spores and Cells in Exponential Growth

PubMed Central

Vold, Barbara S.

1973-01-01

Differences between the transfer ribonucleic acid (tRNA) of spores and exponentially growing cells of Bacillus subtilis 168 were compared by co-chromatography on reversed-phase column RPC-5. This system gave excellent resolution of isoaccepting species in 1 to 2 hr using a 200-ml gradient. Two methods were used to extract spore tRNAs, a procedure using a Braun homogenizer and a pretreatment with dithiothreitol followed by lysis with lysozyme. Where changes were observed, column elution profiles of spore tRNAs were independent of the extraction method used. Three kinds of changes between the profiles of vegetative cell tRNA and spore tRNA were observed: (i) no change; phe-, val-, ala-, asp-, ileu-, pro-, met-, fmet-, and his-tRNAs, (ii) a change in the ratio of existing peaks; gly-, tyr-, leu-, ser-, thr-, aspn-, and arg-tRNAs, and (iii) the appearance or disappearance of unique peaks; lys-, glu-, and trp-tRNAs. PMID:4632322

Assessing the cleanliness of surfaces: Innovative molecular approaches vs. standard spore assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cooper, M.; Duc, M.T. La; Probst, A.

2011-04-01

A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarilymore » give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces.« less

Use of fatty acid methyl ester profiles for discrimination of Bacillus cereus T-strain spores grown on different media.

PubMed

Ehrhardt, Christopher J; Chu, Vivian; Brown, TeeCie; Simmons, Terrie L; Swan, Brandon K; Bannan, Jason; Robertson, James M

2010-03-01

The goal of this study was to determine if cellular fatty acid methyl ester (FAME) profiling could be used to distinguish among spore samples from a single species (Bacillus cereus T strain) that were prepared on 10 different medium formulations. To analyze profile differences and identify FAME biomarkers diagnostic for the chemical constituents in each sporulation medium, a variety of statistical techniques were used, including nonmetric multidimensional scaling (nMDS), analysis of similarities (ANOSIM), and discriminant function analysis (DFA). The results showed that one FAME biomarker, oleic acid (18:1 omega9c), was exclusively associated with spores grown on Columbia agar supplemented with sheep blood and was indicative of blood supplements that were present in the sporulation medium. For spores grown in other formulations, multivariate comparisons across several FAME biomarkers were required to discern profile differences. Clustering patterns in nMDS plots and R values from ANOSIM revealed that dissimilarities among FAME profiles were most pronounced when spores grown with disparate sources of complex additives or protein supplements were compared (R > 0.8), although other factors also contributed to FAME differences. DFA indicated that differentiation could be maximized with a targeted subset of FAME variables, and the relative contributions of branched FAME biomarkers to group dissimilarities changed when different media were compared. When taken together, these analyses indicate that B. cereus spore samples grown in different media can be resolved with FAME profiling and that this may be a useful technique for providing intelligence about the production methods of Bacillus organisms in a forensic investigation.

Use of Fatty Acid Methyl Ester Profiles for Discrimination of Bacillus cereus T-Strain Spores Grown on Different Media▿

PubMed Central

Ehrhardt, Christopher J.; Chu, Vivian; Brown, TeeCie; Simmons, Terrie L.; Swan, Brandon K.; Bannan, Jason; Robertson, James M.

2010-01-01

The goal of this study was to determine if cellular fatty acid methyl ester (FAME) profiling could be used to distinguish among spore samples from a single species (Bacillus cereus T strain) that were prepared on 10 different medium formulations. To analyze profile differences and identify FAME biomarkers diagnostic for the chemical constituents in each sporulation medium, a variety of statistical techniques were used, including nonmetric multidimensional scaling (nMDS), analysis of similarities (ANOSIM), and discriminant function analysis (DFA). The results showed that one FAME biomarker, oleic acid (18:1 ω9c), was exclusively associated with spores grown on Columbia agar supplemented with sheep blood and was indicative of blood supplements that were present in the sporulation medium. For spores grown in other formulations, multivariate comparisons across several FAME biomarkers were required to discern profile differences. Clustering patterns in nMDS plots and R values from ANOSIM revealed that dissimilarities among FAME profiles were most pronounced when spores grown with disparate sources of complex additives or protein supplements were compared (R > 0.8), although other factors also contributed to FAME differences. DFA indicated that differentiation could be maximized with a targeted subset of FAME variables, and the relative contributions of branched FAME biomarkers to group dissimilarities changed when different media were compared. When taken together, these analyses indicate that B. cereus spore samples grown in different media can be resolved with FAME profiling and that this may be a useful technique for providing intelligence about the production methods of Bacillus organisms in a forensic investigation. PMID:20097814

Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis.

PubMed

Delvecchio, Vito G; Connolly, Joseph P; Alefantis, Timothy G; Walz, Alexander; Quan, Marian A; Patra, Guy; Ashton, John M; Whittington, Jessica T; Chafin, Ryan D; Liang, Xudong; Grewal, Paul; Khan, Akbar S; Mujer, Cesar V

2006-09-01

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Delta-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.

Proteomic Profiling and Identification of Immunodominant Spore Antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis‡

PubMed Central

DelVecchio, Vito G.; Connolly, Joseph P.; Alefantis, Timothy G.; Walz, Alexander; Quan, Marian A.; Patra, Guy; Ashton, John M.; Whittington, Jessica T.; Chafin, Ryan D.; Liang, Xudong; Grewal, Paul; Khan, Akbar S.; Mujer, Cesar V.

2006-01-01

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Δ-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development. PMID:16957262

A Computational Approach to Estimating Nondisjunction Frequency in Saccharomyces cerevisiae

PubMed Central

Chu, Daniel B.; Burgess, Sean M.

2016-01-01

Errors segregating homologous chromosomes during meiosis result in aneuploid gametes and are the largest contributing factor to birth defects and spontaneous abortions in humans. Saccharomyces cerevisiae has long served as a model organism for studying the gene network supporting normal chromosome segregation. Measuring homolog nondisjunction frequencies is laborious, and involves dissecting thousands of tetrads to detect missegregation of individually marked chromosomes. Here we describe a computational method (TetFit) to estimate the relative contributions of meiosis I nondisjunction and random-spore death to spore inviability in wild type and mutant strains. These values are based on finding the best-fit distribution of 4, 3, 2, 1, and 0 viable-spore tetrads to an observed distribution. Using TetFit, we found that meiosis I nondisjunction is an intrinsic component of spore inviability in wild-type strains. We show proof-of-principle that the calculated average meiosis I nondisjunction frequency determined by TetFit closely matches empirically determined values in mutant strains. Using these published data sets, TetFit uncovered two classes of mutants: Class A mutants skew toward increased nondisjunction death, and include those with known defects in establishing pairing, recombination, and/or synapsis of homologous chromosomes. Class B mutants skew toward random spore death, and include those with defects in sister-chromatid cohesion and centromere function. Epistasis analysis using TetFit is facilitated by the low numbers of tetrads (as few as 200) required to compare the contributions to spore death in different mutant backgrounds. TetFit analysis does not require any special strain construction, and can be applied to previously observed tetrad distributions. PMID:26747203

Survival of bacterial isolates exposed to simulated Jovian trapped radiation belt electrons and solar wind protons

NASA Technical Reports Server (NTRS)

Taylor, D. M.; Hagen, C. A.; Renninger, G. M.; Simko, G. J.; Smith, C. D.; Yelinek, J. A.

1973-01-01

With missions to Jupiter, the spacecraft will be exposed for extended durations to solar wind radiation and the Jovian trapped radiation belt. This study is designed to determine the effect of these radiation environments on spacecraft bacterial isolates. The information can be used in the probability of contamination analysis for these missions. A bacterial subpopulation from Mariner Mars 1971 spacecraft (nine spore-forming and three non-spore-forming isolates) plus two comparative organisms, Staphylococcus epidermidis ATCC 17917 and a strain of Bacillus subtilis var. niger, were exposed to 2, 12, and 25 MeV electrons at different doses with simultaneous exposure to a vacuum of 1.3 x 10(-4) N m-2 at 20 and -20 degrees C. The radioresistance of the subpopulation was dependent on the isolate, dose and energy of electrons. Temperature affected the radioresistance of only the spore-forming isolates. Survival data indicated that spores were reduced approximately 1 log/1500 J kg-1 (10 J kg-1=1 krad), while non-spore-forming isolates (micrococci) were reduced 1.5-2 logs/1500 J kg-1 with the exception of an apparent radioresistant isolate whose resistance approached that of the spores. The subpopulation was found to be less resistant to lower energy than to higher energy electrons. The bacterial isolates were exposed to 3 keV protons under the same conditions as the electrons with a total fluence of 1.5 x 10(13) p cm-2 and a dose rate of 8.6 x 10(9) p cm-2 s-1. The results showed that only 20% of S. epidermidis and 45% of B. subtilis populations survived exposure to the 3 keV protons, while the mean survival of the spacecraft subpopulation was 45% with a range from 31.8% (non-spore-former) to 64.8% (non-spore-former). No significant difference existed between spore-forming and non-spore-forming isolates.

The aluminium and iodine pentoxide reaction for the destruction of spore forming bacteria.

PubMed

Clark, Billy R; Pantoya, Michelle L

2010-10-21

The threat of biological weapons is a major concern in the present day and has led to studying methods to neutralize spore forming bacteria. A new technique involves the use of a thermite reaction that exhibits biocidal properties to limit bacterial growth. The objective was to examine the influence on bacteria growth upon spore exposure to thermite reactions with and without biocidal properties. Three thermites are considered: two that have biocidal properties (aluminium (Al) combined with iodine pentoxide (I(2)O(5)) and Al combined with silver oxide (Ag(2)O)); and, one that produces a highly exothermic reaction but has no biocidal properties (Al combined with iron oxide (Fe(2)O(3))). Results show that Al + I(2)O(5) is extremely effective at neutralizing spores after only one hour of exposure. The temperature generated by the reaction was not determined to be an influential factor affecting spore growth kinetics. Further analysis of the thermite reactions revealed that the Al + I(2)O(5) reaction produces iodine gas that effectively interacts with the spores and neutralizes bacteria growth, while the Al + Ag(2)O reaction temperature does not vaporize silver. In the condensed phase silver does not interact with the spores enough to neutralize bacteria growth. This study gives evidence that a thermite can be used as a stable transportation and delivery system for biocidal gas.

Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system

PubMed Central

Zhang, Kang; Duan, Xuguo; Wu, Jing

2016-01-01

Bacillus subtilis ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a Streptococcus pyogenes CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA, cas9, and a homologous repair template was established for highly efficient gene disruption in B. subtilis ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the srfC, spoIIAC, nprE, aprE and amyE genes of B. subtilis ATCC 6051a, which hamper its use in industrial fermentation. Compared with B. subtilis ATCC 6051a, the final mutant, BS5 (ΔsrfC, ΔspoIIAC, ΔnprE, ΔaprE, ΔamyE), produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain. PMID:27305971

Extrachromosomal inheritance in Schizosaccharomyces pombe. I. Evidence for an extrakaryotically inherited mutation conferring resistance to antimycin.

PubMed

Wolf, K; Burger, G; Lang, B; Kaudewitz, F

1976-02-27

In crosses of [ANTr8] with auxotrophic strains, resistance to antimycin segregates almost 50:50 in random spore analysis with a slight preponderance for the sensitivity allele. Tetrad analysis, however, shows all possible types of tetrads (2:2; 3:1; 1:3; 4:0; 0:4 resistant versus sensitive) with an excess of 2:2 segregations and sectoring of colonies on antimycin medium indicating an extrachromosomal mode of inheritance. The overall ratio of resistant versus sensitive spores is the same as compared with random spore data. Using a mutant blocked in meiosis (mei 1) mitotic segregation of stable diploids is achieved, leading to a ratio of 20% resistant to 80% sensitive clones. Possible reasons for the bias in transmission of the resistance determinant is discussed.

ANALYSIS OF FUNGAL SPORE MYCOTOXIN AND THE RELATIONSHIP BETWEEN SPORE SURFACE AREA AND MYCOTOXIN CONTENT UTILIZING A PROTEIN TRANSLATION INHIBITION ASSAY

EPA Science Inventory

Due to mounting evidence suggesting that biological contamination in the built environment may cause a myriad of adverse health effects, research aimed at understanding the potential exposure to fungal organisms and their metabolites is of utmost importance. To this end we utiliz...

Survivability of bare, individual Bacillus subtilis spores to high-velocity surface impact: Implications for microbial transfer through space

NASA Astrophysics Data System (ADS)

Barney, Brandon L.; Pratt, Sara N.; Austin, Daniel E.

2016-06-01

Laboratory experiments show that endospores of Bacillus subtilis survive impact against a solid surface at velocities as high as 299 ±28 m/s. During impact, spores experience and survive accelerations of at least 1010 m/s2. The spores were introduced into a vacuum chamber using an electrospray source and accelerated to a narrow velocity distribution by entrainment in a differentially pumped gas flow. Different velocity ranges were studied by modifying the gas flow parameters. The spores were electrically charged, allowing direct measurement of the velocity of each spore as it passed through an image charge detector prior to surface impact. Spores impacted a glass surface and were collected for subsequent analysis by culturing. Most spores survived impact at all measured velocities. These experiments differ fundamentally from other studies that show either shock or impact survivability of bacteria embedded within or on the surface of a projectile. Bacteria in the present experiments undergo a single interaction with a solid surface at the full impact velocity, in the absence of any other effects such as cushioning due to microbe agglomerations, deceleration due to air or vapor, or transfer of impact shock through solid or liquid media. During these full-velocity impact events, the spores experience extremely high decelerations. This study is the first reported instance of accelerations of this magnitude experienced during a bacteria impact event. These results are discussed in the context of potential transfer of viable microbes in space and other scenarios involving surface impacts at high velocities.

Sensitive, Rapid Detection of Bacterial Spores

NASA Technical Reports Server (NTRS)

Kern, Roger G.; Venkateswaran, Kasthuri; Chen, Fei; Pickett, Molly; Matsuyama, Asahi

2009-01-01

A method of sensitive detection of bacterial spores within delays of no more than a few hours has been developed to provide an alternative to a prior three-day NASA standard culture-based assay. A capability for relatively rapid detection of bacterial spores would be beneficial for many endeavors, a few examples being agriculture, medicine, public health, defense against biowarfare, water supply, sanitation, hygiene, and the food-packaging and medical-equipment industries. The method involves the use of a commercial rapid microbial detection system (RMDS) that utilizes a combination of membrane filtration, adenosine triphosphate (ATP) bioluminescence chemistry, and analysis of luminescence images detected by a charge-coupled-device camera. This RMDS has been demonstrated to be highly sensitive in enumerating microbes (it can detect as little as one colony-forming unit per sample) and has been found to yield data in excellent correlation with those of culture-based methods. What makes the present method necessary is that the specific RMDS and the original protocols for its use are not designed for discriminating between bacterial spores and other microbes. In this method, a heat-shock procedure is added prior to an incubation procedure that is specified in the original RMDS protocols. In this heat-shock procedure (which was also described in a prior NASA Tech Briefs article on enumerating sporeforming bacteria), a sample is exposed to a temperature of 80 C for 15 minutes. Spores can survive the heat shock, but nonspore- forming bacteria and spore-forming bacteria that are not in spore form cannot survive. Therefore, any colonies that grow during incubation after the heat shock are deemed to have originated as spores.

Viable spore counts in biological controls pre-sterilization.

PubMed

Brusca, María I; Bernat, María I; Turcot, Liliana; Nastri, Natalia; Nastri, Maria; Rosa, Alcira

2005-01-01

The aim of the present study was to evaluate the total count of viable spores in standardized inoculated carriers pre-sterilization. Samples of "Bacterial Spore Sterilization Strip" (R Biological Laboratories) (well before their expiry date) were divided into Group A (B. subtilis) and Group B (B. stearothermophylus). Twenty-four strips were tested per group. The strips were minced in groups of three, placed in chilled sterile water and vortexed for 5 minutes to obtain a homogenous suspension. Ten ml of the homogenous suspension were transferred to two sterile jars, i.e. one jar per group. The samples were then heated in a water bath at 95 degrees C (Group A) or 80 degrees C (Group B) for 15 minutes and cooled rapidly in an ice bath at 0- 4 degrees C during 15 minutes. Successive dilutions were performed until a final aliquot of 30 to 300 colony-forming units (CFU) was obtained. The inoculums were placed in Petri dishes with culture medium (soy extract, casein agar adapted for spores, melted and cooled to 45-50 degrees C) and incubated at 55 degrees C or 37 degrees C. Statistical analysis of the data was performed. A larger number of spores were found at 48 hours than at 24 hours. However, this finding did not hold true for all the groups. The present results show that monitoring viable spores pre-sterilization would guarantee the accuracy of the data. Total spore counts must be within 50 and 300% of the number of spores indicated in the biological control. The procedure is essential to guarantee the efficacy of the biological control.

The role of fungal proteinases in pathophysiology of Stachybotrys chartarum.

PubMed

Yike, Iwona; Rand, Thomas; Dearborn, Dorr G

2007-10-01

The adverse health effects of Stachybotrys chartarum have often been linked to exposure to the trichothecene mycotoxins. Recent studies have shown that in addition to mycotoxins this fungus is capable of producing and secreting in vivo proteins such as hemolysins and proteinases. Spore extracts obtained from a high trichothecene producing isolate JS 58-17 exhibited a significantly lower proteolytic activity compared to the low trichothecene producer, JS 58-06. Growing isolates on rice or potato dextrose agar results in higher proteolytic activity of the spores compared to those grown on drywall. Proteinases in the spore extracts can hydrolyze gelatin and collagen I and IV. Analysis of zymograms shows the presence of several proteins with proteolytic activity in the spores of S. chartarum. Human tracheal epithelial cells exposed to spore extracts produced significantly higher levels of IL-6, IL-8, and TNF-alpha than control cells. This stimulation of cytokine production was completely abolished by Pefabloc, a serine protease inhibitor. Neutrophil numbers and proinflammatory cytokine (IL1-beta and TNF-alpha) concentrations were highly elevated in the lungs of 7 day old rat pups exposed intratracheally to 4 x 10(4) spores/gm body weight compared to control. No significant differences in those inflammatory indices in vivo were noted between the treatments with the high trichothecene producer, isolate JS 58-17 and JS 58-06, which does not produce macrocyclic trichothecenes. Immunohistochemistry revealed reduced collagen IV labeling in spore-induced lung granulomas in rat pups exposed to both isolates. These results suggest that proteinases from S. chartarum spores significantly contribute to lung inflammation and injury.

Germination and amplification of anthrax spores by soil-dwelling amoebas.

PubMed

Dey, Rafik; Hoffman, Paul S; Glomski, Ian J

2012-11-01

While anthrax is typically associated with bioterrorism, in many parts of the world the anthrax bacillus (Bacillus anthracis) is endemic in soils, where it causes sporadic disease in livestock. These soils are typically rich in organic matter and calcium that promote survival of resilient B. anthracis spores. Outbreaks of anthrax tend to occur in warm weather following rains that are believed to concentrate spores in low-lying areas where runoff collects. It has been concluded that elevated spore concentrations are not the result of vegetative growth as B. anthracis competes poorly against indigenous bacteria. Here, we test an alternative hypothesis in which amoebas, common in moist soils and pools of standing water, serve as amplifiers of B. anthracis spores by enabling germination and intracellular multiplication. Under simulated environmental conditions, we show that B. anthracis germinates and multiplies within Acanthamoeba castellanii. The growth kinetics of a fully virulent B. anthracis Ames strain (containing both the pX01 and pX02 virulence plasmids) and vaccine strain Sterne (containing only pX01) inoculated as spores in coculture with A. castellanii showed a nearly 50-fold increase in spore numbers after 72 h. In contrast, the plasmidless strain 9131 showed little growth, demonstrating that plasmid pX01 is essential for growth within A. castellanii. Electron and time-lapse fluorescence microscopy revealed that spores germinate within amoebal phagosomes, vegetative bacilli undergo multiplication, and, following demise of the amoebas, bacilli sporulate in the extracellular milieu. This analysis supports our hypothesis that amoebas contribute to the persistence and amplification of B. anthracis in natural environments.

A zebrafish larval model reveals early tissue-specific innate immune responses to Mucor circinelloides.

PubMed

Voelz, Kerstin; Gratacap, Remi L; Wheeler, Robert T

2015-11-01

Mucormycosis is an emerging fungal infection that is clinically difficult to manage, with increasing incidence and extremely high mortality rates. Individuals with diabetes, suppressed immunity or traumatic injury are at increased risk of developing disease. These individuals often present with defects in phagocytic effector cell function. Research using mammalian models and phagocytic effector cell lines has attempted to decipher the importance of the innate immune system in host defence against mucormycosis. However, these model systems have not been satisfactory for direct analysis of the interaction between innate immune effector cells and infectious sporangiospores in vivo. Here, we report the first real-time in vivo analysis of the early innate immune response to mucormycete infection using a whole-animal zebrafish larval model system. We identified differential host susceptibility, dependent on the site of infection (hindbrain ventricle and swim bladder), as well as differential functions of the two major phagocyte effector cell types in response to viable and non-viable spores. Larval susceptibility to mucormycete spore infection was increased upon immunosuppressant treatment. We showed for the first time that macrophages and neutrophils were readily recruited in vivo to the site of infection in an intact host and that spore phagocytosis can be observed in real-time in vivo. While exploring innate immune effector recruitment dynamics, we discovered the formation of phagocyte clusters in response to fungal spores that potentially play a role in fungal spore dissemination. Spores failed to activate pro-inflammatory gene expression by 6 h post-infection in both infection models. After 24 h, induction of a pro-inflammatory response was observed only in hindbrain ventricle infections. Only a weak pro-inflammatory response was initiated after spore injection into the swim bladder during the same time frame. In the future, the zebrafish larva as a live whole-animal model system will contribute greatly to the study of molecular mechanisms involved in the interaction of the host innate immune system with fungal spores during mucormycosis. © 2015. Published by The Company of Biologists Ltd.

Dry-heat Resistance of Bacillus Subtilis Var. Niger Spores on Mated Surfaces

NASA Technical Reports Server (NTRS)

Simko, G. J.; Devlin, J. D.; Wardle, M. D.

1971-01-01

Bacillus subtilis var. niger spores were placed on the surfaces of test coupons manufactured from typical spacecraft materials including stainless steel, magnesium, titanium, and aluminum. These coupons were then juxtaposed at the inoculated surfaces and subjected to test pressures of 0, 1000, 5000, and 10,000 psi. Tests were conducted in ambient, nitrogen, and helium atmospheres. While under the test pressure condition, the spores were exposed to 125 C for intervals of 5, 10, 20, 50, or 80 min. Survivor data were subjected to a linear regression analysis that calculated decimal reduction times.

Effects of steam autoclave treatment on Geobacillus stearothermophilus spores.

PubMed

Huesca-Espitia, L C; Suvira, M; Rosenbeck, K; Korza, G; Setlow, B; Li, W; Wang, S; Li, Y-Q; Setlow, P

2016-11-01

To determine the mechanism of autoclave killing of Geobacillus stearothermophilus spores used in biological indicators (BIs) for steam autoclave sterilization, and rates of loss of spore viability and a spore enzyme used in BIs. Spore viability, dipicolinic acid (DPA) release, nucleic acid staining, α-glucosidase activity, protein structure and mutagenesis were measured during autoclaving of G. stearothermophilus spores. Loss of DPA and increases in spore core nucleic acid staining were slower than loss of spore viability. Spore core α-glucosidase was also lost more slowly than spore viability, although soluble α-glucosidase in spore preparations was lost more rapidly. However, spores exposed to an effective autoclave sterilization lost all viability and α-glucosidase activity. Apparently killed autoclaved spores were not recovered by artificial germination in supportive media, much spore protein was denatured during autoclaving, and partially killed autoclave-treated spore preparations did not acquire mutations. These results indicate that autoclave-killed spores cannot be revived, spore killing by autoclaving is likely by protein damage, and spore core α-glucosidase activity is lost more slowly than spore viability. This work provides insight into the mechanism of autoclave killing of spores of an organism used in BIs, and that a spore enzyme in a BI is more stable to autoclaving than spore viability. © 2016 The Society for Applied Microbiology.

Retrospective Species Identification of Microsporidian Spores in Diarrheic Fecal Samples from Human Immunodeficiency Virus/AIDS Patients by Multiplexed Fluorescence In Situ Hybridization▿

PubMed Central

Graczyk, Thaddeus K.; Johansson, Michael A.; Tamang, Leena; Visvesvara, Govinda S.; Moura, Laci S.; DaSilva, Alexandre J.; Girouard, Autumn S.; Matos, Olga

2007-01-01

In order to assess the applicability of multiplexed fluorescence in situ hybridization (FISH) assay for the clinical setting, we conducted retrospective analysis of 110 formalin-stored diarrheic stool samples from human immunodeficiency virus (HIV)/AIDS patients with intestinal microsporidiosis collected between 1992 and 2003. The multiplexed FISH assay identified microsporidian spores in 94 of 110 (85.5%) samples: 49 (52.1%) were positive for Enterocytozoon bieneusi, 43 (45.8%) were positive for Encephalitozoon intestinalis, 2 (2.1%) were positive for Encephalitozoon hellem, and 9 samples (9.6%) contained both E. bieneusi and E. intestinalis spores. Quantitative spore counts per ml of stool yielded concentration values from 3.5 × 103 to 4.4 × 105 for E. bieneusi (mean, 8.8 × 104/ml), 2.3 × 102 to 7.8 × 104 (mean, 1.5 × 104/ml) for E. intestinalis, and 1.8 × 102 to 3.6 × 102 for E. hellem (mean, 2.7 × 102/ml). Identification of microsporidian spores by multiplex FISH assay was more sensitive than both Chromotrope-2R and CalcoFluor White M2R stains; 85.5% versus 72.7 and 70.9%, respectively. The study demonstrated that microsporidian coinfection in HIV/AIDS patients with intestinal microsporidiosis is not uncommon and that formalin-stored fecal samples older than 10 years may not be suitable for retrospective analysis by techniques targeting rRNA. Multiplexed FISH assay is a reliable, quantitative fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, and E. hellem, as well as Encephalitozoon cuniculi, spores in fecal samples and is a useful tool for assessing spore shedding intensity in intestinal microsporidiosis. The method can be used for epidemiological investigations and applied in clinical settings. PMID:17287331

Analysis of atmospheric paniculate matter; application of optical and selected geochemical techniques

USGS Publications Warehouse

Mastalerz, Maria; Glikson, M.; Simpson, R.W.

1999-01-01

An increase in participate matter in the atmosphere has been shown to be linked to increased mortality but this relationship is poorly understood. Light microscopy, electron microscopy, electron microprobe, and micro-FTIR techniques have been applied to study atmospheric particulates in Brisbane, Australia as a part of a study on asthma. The particulate matter samples were collected daily from April to August 1992, and the sampling covered the autumn period which is typically a time of high asthma incidence in Brisbane. Volumetrically, most atmospheric particulate matter is less than 2 ??m in size. The microscopic analysis reveals that this material is composed mainly of combusted and incompletely burned hydrocarbons from motor vehicle exhaust emissions, quiescent spores of Mucorales, soil bacteria, and inorganic matter in the form of quartz and other silicates. Elemental and functional group analyses confirm microscope identification, documenting carbon-rich, aromatic exhaust material, more aliphatic pollen and spore material and inorganic matter. Fungal spores dominate bioaerosol and are very abundant from the end of April through May to mid-June. The cytoplasmic content of pollens or fungal spores is commonly regarded as allergenic. Particulates from the exhaust emissions and crustal material in a sub-micrometer size range may act as carriers or dispersive mechanisms for cytoplasmic material from fungal spores and pollens, perhaps causing periods of the highest exhaust emission to be the most allergenic. ?? 1998 Elsevier Science B.V. All rights reserved.

Airborne myxomycete spores: detection using molecular techniques

NASA Astrophysics Data System (ADS)

Kamono, Akiko; Kojima, Hisaya; Matsumoto, Jun; Kawamura, Kimitaka; Fukui, Manabu

2009-01-01

Myxomycetes are organisms characterized by a life cycle that includes a fruiting body stage. Myxomycete fruiting bodies contain spores, and wind dispersal of the spores is considered important for this organism to colonize new areas. In this study, the presence of airborne myxomycetes and the temporal changes in the myxomycete composition of atmospheric particles (aerosols) were investigated with a polymerase chain reaction (PCR)-based method for Didymiaceae and Physaraceae. Twenty-one aerosol samples were collected on the roof of a three-story building located in Sapporo, Hokkaido Island, northern Japan. PCR analysis of DNA extracts from the aerosol samples indicated the presence of airborne myxomycetes in all the samples, except for the one collected during the snowfall season. Denaturing gradient gel electrophoresis (DGGE) analysis of the PCR products showed seasonally varying banding patterns. The detected DGGE bands were subjected to sequence analyses, and four out of nine obtained sequences were identical to those of fruiting body samples collected in Hokkaido Island. It appears that the difference in the fruiting period of each species was correlated with the seasonal changes in the myxomycete composition of the aerosols. Molecular evidence shows that newly formed spores are released and dispersed in the air, suggesting that wind-driven dispersal of spores is an important process in the life history of myxomycetes. This study is the first to detect airborne myxomycetes with the use of molecular ecological analyses and to characterize their seasonal distribution.

Genotyping of single spore isolates of a Pasteuria penetrans population occurring in Florida using SNP-based markers.

PubMed

Joseph, S; Schmidt, L M; Danquah, W B; Timper, P; Mekete, T

2017-02-01

To generate single spore lines of a population of bacterial parasite of root-knot nematode (RKN), Pasteuria penetrans, isolated from Florida and examine genotypic variation and virulence characteristics exist within the population. Six single spore lines (SSP), 16SSP, 17SSP, 18SSP, 25SSP, 26SSP and 30SSP were generated. Genetic variability was evaluated by comparing single-nucleotide polymorphisms (SNPs) in six protein-coding genes and the 16S rRNA gene. An average of one SNP was observed for every 69 bp in the 16S rRNA, whereas no SNPs were observed in the protein-coding sequences. Hierarchical cluster analysis of 16S rRNA sequences placed the clones into three distinct clades. Bio-efficacy analysis revealed significant heterogeneity in the level virulence and host specificity between the individual clones. The SNP markers developed to the 5' hypervariable region of the 16S rRNA gene may be useful in biotype differentiation within a population of P. penetrans. This study demonstrates an efficient method for generating single spore lines of P. penetrans and gives a deep insight into genetic heterogeneity and varying level of virulence exists within a population parasitizing a specific Meloidogyne sp. host. The results also suggest that the application of generalist spore lines in nematode management may achieve broad RKN control. © 2016 The Society for Applied Microbiology.

Alternative Sigma Factors SigF, SigE, and SigG Are Essential for Sporulation in Clostridium botulinum ATCC 3502

PubMed Central

Kirk, David G.; Zhang, Zhen; Korkeala, Hannu

2014-01-01

Clostridium botulinum produces heat-resistant endospores that may germinate and outgrow into neurotoxic cultures in foods. Sporulation is regulated by the transcription factor Spo0A and the alternative sigma factors SigF, SigE, SigG, and SigK in most spore formers studied to date. We constructed mutants of sigF, sigE, and sigG in C. botulinum ATCC 3502 and used quantitative reverse transcriptase PCR and electron microscopy to assess their expression of the sporulation pathway on transcriptional and morphological levels. In all three mutants the expression of spo0A was disrupted. The sigF and sigE mutants failed to induce sigG and sigK beyond exponential-phase levels and halted sporulation during asymmetric cell division. In the sigG mutant, peak transcription of sigE was delayed and sigK levels remained lower than that in the parent strain. The sigG mutant forespore was engulfed by the mother cell and possessed a spore coat but no peptidoglycan cortex. The findings suggest that SigF and SigE of C. botulinum ATCC 3502 are essential for early sporulation and late-stage induction of sigK, whereas SigG is essential for spore cortex formation but not for coat formation, as opposed to previous observations in B. subtilis sigG mutants. Our findings add to a growing body of evidence that regulation of sporulation in C. botulinum ATCC 3502, and among the clostridia, differs from the B. subtilis model. PMID:24928875

Sporulation and germination gene expression analysis of Bacillus anthracis Sterne spores in skim milk under heat and different intervention techniques

USDA-ARS?s Scientific Manuscript database

To investigate how B. anthracis Stene spores survive in milk under heat (80 degree C, 10 minutes), pasteurization (72 degree C, 15 seconds) and pasteurization plus microfiltration, the expression levels of genes that related to sporulation and germination were tested using real-time PCR assays. Tw...

Bacillus odysseyi sp. nov., a round-spore-forming bacillus isolated from the Mars Odyssey spacecraft

NASA Technical Reports Server (NTRS)

La Duc, Myron T.; Satomi, Masataka; Venkateswaran, Kasthuri

2004-01-01

A round-spore-forming Bacillus species that produces an exosporium was isolated from the surface of the Mars Odyssey spacecraft. This novel species has been characterized on the basis of phenotypic traits, 16S rDNA sequence analysis and DNA-DNA hybridization. According to the results of these analyses, this strain belongs to the genus Bacillus and is a Gram-positive, aerobic, rod-shaped, endospore-forming eubacterium. Ultrathin sections of the spores showed the presence of an exosporium, spore coat, cortex and core. 16S rDNA sequence similarities between this strain, Bacillus fusiformis and Bacillus silvestris were approximately 96% and DNA-DNA reassociation values with these two bacilli were 23 and 17%, respectively. Spores of the novel species were resistant to desiccation, H2O2 and UV and gamma radiation. Of all strains tested, the spores of this strain were the most consistently resistant and survived all of the challenges posed, i.e. exposure to conditions of desiccation (100% survival), H2O2 (26% survival), UV radiation (10% survival at 660 J m(-2)) and gamma radiation (0.4% survival). The name proposed for this novel bacterium is Bacillus odysseyi sp. nov.; the type strain is 34hs-1T (=ATCC PTA-4993T=NRRL B-30641T=NBRC 100172T).

Kudoa prunusi n. sp. (Myxozoa: Multivalvulida) from the brain of Pacific bluefin tuna Thunnus orientalis (Temminck & Schlegel, 1844) cultured in Japan.

PubMed

Meng, F; Yokoyama, H; Shirakashi, S; Grabner, D; Ogawa, K; Ishimaru, K; Sawada, Y; Murata, O

2011-01-01

Kudoa prunusi n. sp. (Myxozoa; Multivalvulida) is described from the brain of Pacific bluefin tuna Thunnus orientalis cultured in Japan. Numerous white cysts, up to 0.5mm in size, were found on and in the brain. Spores having typically five spore valves and five polar capsules resembled a five-petal cherry blossom in apical view and were conical shape with a round bottom in side view. Average spore size was 9.63 (8.5-10.3) μm in width and 7.50 (6.7-8.6) μm in length. The spore dimensions of K. prunusi overlapped with those of Kudoa yasunagai ex Sillago ciliata having five to six spore valves, but they were clearly distinct in spore shape, 18S rDNA and 28S rDNA sequences (0.3% and 1.7% differences, respectively). Phylogenetic analysis of 18S rDNA revealed that K. prunusi grouped with the brain-infecting multivalvulid species, K. yasunagai, K. chaetodoni, K. lethrini and K. neurophila, rather than five-valved Kudoa spp. Combined with morphological, molecular and biological differences, K. prunusi was proven to be a new species. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

PsB multiprotein complex of Dictyostelium discoideum. Demonstration of cellulose binding activity and order of protein subunit assembly.

PubMed

McGuire, V; Alexander, S

1996-06-14

The differentiated spores of Dictyostelium are surrounded by an extracellular matrix, the spore coat, which protects them from environmental factors allowing them to remain viable for extended periods of time. This presumably is a major evolutionary advantage. This unique extracellular matrix is composed of cellulose and glycoproteins. Previous work has shown that some of these spore coat glycoproteins exist as a preassembled multiprotein complex (the PsB multiprotein complex) which is stored in the prespore vesicles (Watson, N., McGuire, V., and Alexander, S (1994) J. Cell Sci. 107, 2567-2579). Later in development, the complex is synchronously secreted from the prespore vesicles and incorporated into the spore coat. We now have shown that the PsB complex has a specific in vitro cellulose binding activity. The analysis of mutants lacking individual subunits of the PsB complex revealed the relative order of assembly of the subunit proteins and demonstrated that the protein subunits must be assembled for cellulose binding activity. These results provide a biochemical explanation for the localization of this multiprotein complex in the spore coat.

Trichoderma sp. spores and Kluyveromyces marxianus cells magnetic separation: Immobilization on chitosan-coated magnetic nanoparticles.

PubMed

Palacios-Ponce, Sócrates; Ramos-González, Rodolfo; Ruiz, Héctor A; Aguilar, Miguel A; Martínez-Hernández, José L; Segura-Ceniceros, Elda P; Aguilar, Cristóbal N; Michelena, Georgina; Ilyina, Anna

2017-07-03

In the present study, the interactions between chitosan-coated magnetic nanoparticles (C-MNP) and Trichoderma sp. spores as well as Kluyveromyces marxianus cells were studied. By Plackett-Burman design, it was demonstrated that factors which directly influenced on yeast cell immobilization and magnetic separation were inoculum and C-MNP quantity, stirring speed, interaction time, and volume of medium, while in the case of fungal spores, the temperature also was disclosed as an influencing factor. Langmuir and Freundlich models were applied for the mathematical analysis of adsorption isotherms at 30°C. For Trichoderma sp. spore adsorption isotherm, the highest correlation coefficient was observed for lineal function of Langmuir model with a maximum adsorption capacity at 5.00E + 09 spores (C-MNP g -1 ). Adsorption isotherm of K. marxianus cells was better adjusted to Freundlich model with a constant (K f ) estimated as 2.05E + 08 cells (C-MNP g -1 ). Both systems may have a novel application in fermentation processes assisted with magnetic separation of biomass.

Atg22p, a vacuolar membrane protein involved in the amino acid compartmentalization of Schizosaccharomyces pombe.

PubMed

Sugimoto, Naoko; Iwaki, Tomoko; Chardwiriyapreecha, Soracom; Shimazu, Masamitsu; Kawano, Miyuki; Sekito, Takayuki; Takegawa, Kaoru; Kakinuma, Yoshimi

2011-01-01

The fission yeast Schizosaccharomyces pombe has a homolog of the budding yeast Atg22p, which is involved in spore formation (Mukaiyama H. et al., Microbiology, 155, 3816-3826 (2009)). GFP-tagged Atg22p in the fission yeast was localized to the vacuolar membrane. Upon disruption of atg22, the amino acid levels of the cellular fraction as well as the vacuolar fraction decreased. The uptake of several amino acids, such as lysine, histidine, and arginine, was impaired in atg22Δ cells. S. pombe Atg22p plays an important role in the compartmentalization of amino acids.

Mechanism of Bacillus subtilis spore inactivation by and resistance to supercritical CO2 plus peracetic acid.

PubMed

Setlow, B; Korza, G; Blatt, K M S; Fey, J P; Setlow, P

2016-01-01

Determine how supercritical CO2 (scCO2 ) plus peracetic acid (PAA) inactivates Bacillus subtilis spores, factors important in spore resistance to scCO2 -PAA, and if spores inactivated by scCO2 -PAA are truly dead. Spores of wild-type B. subtilis and isogenic mutants lacking spore protective proteins were treated with scCO2 -PAA in liquid or dry at 35°C. Wild-type wet spores (aqueous suspension) were more susceptible than dry spores. Treated spores were examined for viability (and were truly dead), dipicolinic acid (DPA), mutations, permeability to nucleic acid stains, germination under different conditions, energy metabolism and outgrowth. ScCO2 -PAA-inactivated spores retained DPA, and survivors had no notable DNA damage. However, DPA was released from inactivated spores at a normally innocuous temperature (85°C), and colony formation from treated spores was salt sensitive. The inactivated spores germinated but did not outgrow, and these germinated spores had altered plasma membrane permeability and defective energy metabolism. Wet or dry coat-defective spores had increased scCO2 -PAA sensitivity, and dry spores but not wet spores lacking DNA protective proteins were more scCO2 -PAA sensitive. These findings suggest that scCO2 -PAA inactivates spores by damaging spores' inner membrane. The spore coat provided scCO2 -PAA resistance for both wet and dry spores. DNA protective proteins provided scCO2 -PAA resistance only for dry spores. These results provide information on mechanisms of spore inactivation of and resistance to scCO2 -PAA, an agent with increasing use in sterilization applications. © 2015 The Society for Applied Microbiology.

Survival of Bacillus pumilus spores for a prolonged period of time in real space conditions.

PubMed

Vaishampayan, Parag A; Rabbow, Elke; Horneck, Gerda; Venkateswaran, Kasthuri J

2012-05-01

To prevent forward contamination and maintain the scientific integrity of future life-detection missions, it is important to characterize and attempt to eliminate terrestrial microorganisms associated with exploratory spacecraft and landing vehicles. Among the organisms isolated from spacecraft-associated surfaces, spores of Bacillus pumilus SAFR-032 exhibited unusually high resistance to decontamination techniques such as UV radiation and peroxide treatment. Subsequently, B. pumilus SAFR-032 was flown to the International Space Station (ISS) and exposed to a variety of space conditions via the European Technology Exposure Facility (EuTEF). After 18 months of exposure in the EXPOSE facility of the European Space Agency (ESA) on EuTEF under dark space conditions, SAFR-032 spores showed 10-40% survivability, whereas a survival rate of 85-100% was observed when these spores were kept aboard the ISS under dark simulated martian atmospheric conditions. In contrast, when UV (>110 nm) was applied on SAFR-032 spores for the same time period and under the same conditions used in EXPOSE, a ∼7-log reduction in viability was observed. A parallel experiment was conducted on Earth with identical samples under simulated space conditions. Spores exposed to ground simulations showed less of a reduction in viability when compared with the "real space" exposed spores (∼3-log reduction in viability for "UV-Mars," and ∼4-log reduction in viability for "UV-Space"). A comparative proteomics analysis indicated that proteins conferring resistant traits (superoxide dismutase) were present in higher concentration in space-exposed spores when compared to controls. Also, the first-generation cells and spores derived from space-exposed samples exhibited elevated UVC resistance when compared with their ground control counterparts. The data generated are important for calculating the probability and mechanisms of microbial survival in space conditions and assessing microbial contaminants as risks for forward contamination and in situ life detection.

The Myxococcus xanthus Spore Cuticula Protein C Is a Fragment of FibA, an Extracellular Metalloprotease Produced Exclusively in Aggregated Cells

PubMed Central

Lee, Bongsoo; Mann, Petra; Grover, Vidhi; Treuner-Lange, Anke; Kahnt, Jörg; Higgs, Penelope I.

2011-01-01

Myxococcus xanthus is a soil bacterium with a complex life cycle involving distinct cell fates, including production of environmentally resistant spores to withstand periods of nutrient limitation. Spores are surrounded by an apparently self-assembling cuticula containing at least Proteins S and C; the gene encoding Protein C is unknown. During analyses of cell heterogeneity in M. xanthus, we observed that Protein C accumulated exclusively in cells found in aggregates. Using mass spectrometry analysis of Protein C either isolated from spore cuticula or immunoprecipitated from aggregated cells, we demonstrate that Protein C is actually a proteolytic fragment of the previously identified but functionally elusive zinc metalloprotease, FibA. Subpopulation specific FibA accumulation is not due to transcriptional regulation suggesting post-transcriptional regulation mechanisms mediate its heterogeneous accumulation patterns. PMID:22174937

Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis.

PubMed

Bishop, Alistair H; Gowen, Simon R; Pembroke, Barbara; Trotter, James R

2007-09-01

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).

Mechanism of Bacillus subtilis Spore Inactivation by and Resistance to Supercritical CO2 plus Peracetic Acid

PubMed Central

Setlow, Barbara; Korza, George; Blatt, Kelly M.S.; Fey, Julien P.; Setlow, Peter

2015-01-01

Aims Determine how supercritical CO2 (scCO2) plus peracetic acid (PAA) inactivates Bacillus subtilis spores, factors important in spore resistance to scCO2-PAA, and if spores inactivated by scCO2-PAA are truly dead. Methods and Results Spores of wild-type B. subtilis and isogenic mutants lacking spore protective proteins were treated with scCO2-PAA in liquid or dry at 35°C. Wild-type wet spores (aqueous suspension) were more susceptible than dry spores. Treated spores were examined for viability (and were truly dead), dipicolinic acid (DPA), mutations, permeability to nucleic acid stains, germination under different conditions, energy metabolism and outgrowth. ScCO2-PAA-inactivated spores retained DPA, and survivors had no notable DNA damage. However, DPA was released from inactivated spores at a normally innocuous temperature (85°C), and colony formation from treated spores was salt sensitive. The inactivated spores germinated but did not outgrow, and these germinated spores had altered plasma membrane permeability and defective energy metabolism. Wet or dry coat-defective spores had increased scCO2-PAA sensitivity, and dry spores but not wet spores lacking DNA protective proteins were more scCO2-PAA sensitive. Conclusions These findings suggest that scCO2-PAA inactivates spores by damaging spores’ inner membrane. The spore coat provided scCO2-PAA resistance for both wet and dry spores. DNA protective proteins provided scCO2-PAA resistance only for dry spores. Significance and Impact of Study These results provide information on mechanisms of spore inactivation of and resistance to scCO2-PAA, an agent with increasing use in sterilization applications. PMID:26535794

Size matters for violent discharge height and settling speed of Sphagnum spores: important attributes for dispersal potential.

PubMed

Sundberg, Sebastian

2010-02-01

Initial release height and settling speed of diaspores are biologically controlled components which are key to modelling wind dispersal. Most Sphagnum (peat moss) species have explosive spore liberation. In this study, how capsule and spore sizes affect the height to which spores are propelled were measured, and how spore size and spore number of discharged particles relate to settling speed in the aspherical Sphagnum spores. Spore discharge and spore cloud development were filmed in a closed chamber (nine species). Measurements were taken from snapshots at three stages of cloud development. Settling speed of spores (14 species) and clusters were timed in a glass tube. The maximum discharge speed measured was 3.6 m s(-1). Spores reached a maximum height of 20 cm (average: 15 cm) above the capsule. The cloud dimensions at all stages were related positively to capsule size (R(2) = 0.58-0.65). Thus species with large shoots (because they have large capsules) have a dispersal advantage. Half of the spores were released as singles and the rest as clusters (usually two to four spores). Single spores settled at 0.84-1.86 cm s(-1), about 52 % slower than expected for spherical spores with the same diameters. Settling speed displayed a positive curvilinear relationship with spore size, close to predictions by Stokes' law for spherical spores with 68 % of the actual diameters. Light-coloured spores settled slower than dark spores. Settling speed of spore clusters agrees with earlier studies. Effective spore discharge and small, slowly settling spores appear particularly important for species in forested habitats. The spore discharge heights in Sphagnum are among the greatest for small, wind-dispersed propagules. The discharge heights and the slow settling of spores affect dispersal distances positively and may help to explain the wide distribution of most boreal Sphagnum species.

Size matters for violent discharge height and settling speed of Sphagnum spores: important attributes for dispersal potential

PubMed Central

Sundberg, Sebastian

2010-01-01

Background and Aims Initial release height and settling speed of diaspores are biologically controlled components which are key to modelling wind dispersal. Most Sphagnum (peat moss) species have explosive spore liberation. In this study, how capsule and spore sizes affect the height to which spores are propelled were measured, and how spore size and spore number of discharged particles relate to settling speed in the aspherical Sphagnum spores. Methods Spore discharge and spore cloud development were filmed in a closed chamber (nine species). Measurements were taken from snapshots at three stages of cloud development. Settling speed of spores (14 species) and clusters were timed in a glass tube. Key Results The maximum discharge speed measured was 3·6 m s−1. Spores reached a maximum height of 20 cm (average: 15 cm) above the capsule. The cloud dimensions at all stages were related positively to capsule size (R2 = 0·58–0·65). Thus species with large shoots (because they have large capsules) have a dispersal advantage. Half of the spores were released as singles and the rest as clusters (usually two to four spores). Single spores settled at 0·84–1·86 cm s−1, about 52 % slower than expected for spherical spores with the same diameters. Settling speed displayed a positive curvilinear relationship with spore size, close to predictions by Stokes' law for spherical spores with 68 % of the actual diameters. Light-coloured spores settled slower than dark spores. Settling speed of spore clusters agrees with earlier studies. Effective spore discharge and small, slowly settling spores appear particularly important for species in forested habitats. Conclusions The spore discharge heights in Sphagnum are among the greatest for small, wind-dispersed propagules. The discharge heights and the slow settling of spores affect dispersal distances positively and may help to explain the wide distribution of most boreal Sphagnum species. PMID:20123930

Fungal Spores Viability on the International Space Station

NASA Astrophysics Data System (ADS)

Gomoiu, I.; Chatzitheodoridis, E.; Vadrucci, S.; Walther, I.; Cojoc, R.

2016-11-01

In this study we investigated the security of a spaceflight experiment from two points of view: spreading of dried fungal spores placed on the different wafers and their viability during short and long term missions on the International Space Station (ISS). Microscopic characteristics of spores from dried spores samples were investigated, as well as the morphology of the colonies obtained from spores that survived during mission. The selected fungal species were: Aspergillus niger, Cladosporium herbarum, Ulocladium chartarum, and Basipetospora halophila. They have been chosen mainly based on their involvement in the biodeterioration of different substrate in the ISS as well as their presence as possible contaminants of the ISS. From biological point of view, three of the selected species are black fungi, with high melanin content and therefore highly resistant to space radiation. The visual inspection and analysis of the images taken before and after the short and the long term experiments have shown that all biocontainers were returned to Earth without damages. Microscope images of the lids of the culture plates revealed that the spores of all species were actually not detached from the surface of the wafers and did not contaminate the lids. From the adhesion point of view all types of wafers can be used in space experiments, with a special comment on the viability in the particular case of iron wafers when used for spores that belong to B. halophila (halophilic strain). This is encouraging in performing experiments with fungi without risking contamination. The spore viability was lower in the experiment for long time to ISS conditions than that of the short experiment. From the observations, it is suggested that the environment of the enclosed biocontainer, as well as the species'specific behaviour have an important effect, reducing the viability in time. Even the spores were not detached from the surface of the wafers, it was observed that spores used in the long term experiment lost the outer layer of their coat without affecting the viability since they were still protected by the middle and the inner layer of the coating. This research highlights a new protocol to perform spaceflight experiments inside the ISS with fungal spores in microgravity conditions, under the additional effect of possible cosmic radiation. According to this protocol the results are expressed in terms of viability, microscopic and morphological changes.

Fungal Spores Viability on the International Space Station.

PubMed

Gomoiu, I; Chatzitheodoridis, E; Vadrucci, S; Walther, I; Cojoc, R

2016-11-01

In this study we investigated the security of a spaceflight experiment from two points of view: spreading of dried fungal spores placed on the different wafers and their viability during short and long term missions on the International Space Station (ISS). Microscopic characteristics of spores from dried spores samples were investigated, as well as the morphology of the colonies obtained from spores that survived during mission. The selected fungal species were: Aspergillus niger, Cladosporium herbarum, Ulocladium chartarum, and Basipetospora halophila. They have been chosen mainly based on their involvement in the biodeterioration of different substrate in the ISS as well as their presence as possible contaminants of the ISS. From biological point of view, three of the selected species are black fungi, with high melanin content and therefore highly resistant to space radiation. The visual inspection and analysis of the images taken before and after the short and the long term experiments have shown that all biocontainers were returned to Earth without damages. Microscope images of the lids of the culture plates revealed that the spores of all species were actually not detached from the surface of the wafers and did not contaminate the lids. From the adhesion point of view all types of wafers can be used in space experiments, with a special comment on the viability in the particular case of iron wafers when used for spores that belong to B. halophila (halophilic strain). This is encouraging in performing experiments with fungi without risking contamination. The spore viability was lower in the experiment for long time to ISS conditions than that of the short experiment. From the observations, it is suggested that the environment of the enclosed biocontainer, as well as the species'specific behaviour have an important effect, reducing the viability in time. Even the spores were not detached from the surface of the wafers, it was observed that spores used in the long term experiment lost the outer layer of their coat without affecting the viability since they were still protected by the middle and the inner layer of the coating. This research highlights a new protocol to perform spaceflight experiments inside the ISS with fungal spores in microgravity conditions, under the additional effect of possible cosmic radiation. According to this protocol the results are expressed in terms of viability, microscopic and morphological changes.

Morphological and genetic characterization of group I Clostridium botulinum type B strain 111 and the transcriptional regulator spoIIID gene knockout mutant in sporulation.

PubMed

Hosomi, Koji; Kuwana, Ritsuko; Takamatsu, Hiromu; Kohda, Tomoko; Kozaki, Shunji; Mukamoto, Masafumi

2015-06-01

Clostridium botulinum is a heat-resistant spore-forming bacterium that causes the serious paralytic illness botulism. Heat-resistant spores may cause food sanitation hazards and sporulation plays a central role in the survival of C. botulinum. We observed morphological changes and investigated the role of the transcriptional regulator SpoIIID in the sporulation of C. botulinum type B strain 111 in order to elucidate the molecular mechanism in C. botulinum. C. botulinum type B formed heat-resistant spores through successive morphological changes corresponding to those of Bacillus subtilis, a spore-forming model organism. An analysis of the spoIIID gene knockout mutant revealed that the transcriptional regulator SpoIIID contributed to heat-resistant spore formation by C. botulinum type B and activated the transcription of the sigK gene later during sporulation. Transcription of the spoIIID gene, which differed from that in B. subtilis and Clostridium difficile, was observed in the sigE gene knockout mutant of C. botulinum type B. An analysis of the sigF gene knockout mutant showed that the sporulation-specific sigma factor SigF was essential for transcription of the spoIIID gene in C. botulinum type B. These results suggest that the regulation of sporulation in C. botulinum is not similar to that in B. subtilis and other clostridia. Copyright © 2015 Elsevier Ltd. All rights reserved.

Relation of indoor and outdoor airborne fungal spore levels in the Kansas City metropolitan area.

PubMed

Jara, David; Portnoy, Jay; Dhar, Minati; Barnes, Charles

2017-03-01

Environmental control is an important component of asthma management for persons with asthma. A damp indoor environment and elevated airborne spore levels are factors in housing environmental control. We investigated if indoor airborne fungal spore levels correlated with outdoor ground-level airborne fungal spores or outdoor centrally collected spore levels as to types and abundance. Air collections were taken from home interiors, outdoor areas adjacent to the homes, and at a central location in the metropolitan area at the approximate same time. All air collections were examined and enumerated microscopically, and airborne spore estimates per cubic meter of air were reported for total fungal spores and for 11 identifiable spore groups. The 244 homes in the study were typical of the North American Midwest. The overall mean total spore counts in spores per cubic meter of air was indoors (4076 spores/m3), outdoors at ground level (8899 spores/m3), and outdoor metropolitan area (8342 spores/m3). All of the major indoor taxa were strongly correlated with the mean total spores present in the home. Total outdoor ground spore levels were highly correlated with levels of major outdoor taxa, such as ascospores and Cladosporium. Correlations of indoor spore levels with outdoor spore levels are strong for most major outdoor taxa. Indoor Aspergillus-Penicillium and Chaetomium are significantly correlated between indoor and local ground-level outdoor air. Although conditions may exist where indoor or outdoor spore levels were not well aligned, in most circumstances, the outdoor airborne spore community was reflected in the indoor airborne spore community.

On the neutralization of bacterial spores in post-detonation flows

NASA Astrophysics Data System (ADS)

Gottiparthi, K. C.; Schulz, J. C.; Menon, S.

2014-09-01

In multiple operational scenarios, explosive charges are used to neutralize confined or unconfined stores of bacterial spores. The spore destruction is achieved by post-detonation combustion and mixing of hot detonation product gases with the ambient flow and spore clouds. In this work, blast wave interaction with bacterial spore clouds and the effect of post-detonation combustion on spore neutralization are investigated using numerical simulations. Spherical explosive charges (radius, = 5.9 cm) comprising of nitromethane are modeled in the vicinity of a spore cloud, and the spore kill in the post-detonation flow is quantified. The effect of the mass of the spores and the initial distance, , of the spore cloud from the explosive charge on the percentage of spores neutralized is investigated. When the spores are initially placed within a distance of 3.0, within 0.1 ms after detonation of the charge, all the spores are neutralized by the blast wave and the hot detonation product gases. In contrast, almost all the spores survived the explosion when is greater than 8.0. The percentage of intact spores varied from 0 to 100 for 3.0 8.0 with spore neutralization dependent on time spent by the spores in the post-detonation mixing/combustion zone.

Antifungal activities of selected essential oils against Fusarium oxysporum f. sp. lycopersici 1322, with emphasis on Syzygium aromaticum essential oil.

PubMed

Sharma, Abhishek; Rajendran, Sasireka; Srivastava, Ankit; Sharma, Satyawati; Kundu, Bishwajit

2017-03-01

The antifungal effects of four essential oils viz., clove (Syzygium aromaticum), lemongrass (Cymbopogon citratus), mint (Mentha × piperita) and eucalyptus (Eucalyptus globulus) were evaluated against wilt causing fungus, Fusarium oxysporum f. sp. lycopersici 1322. The inhibitory effect of oils showed dose-dependent activity on the tested fungus. Most active being the clove oil, exhibiting complete inhibition of mycelial growth and spore germination at 125 ppm with IC 50 value of 18.2 and 0.3 ppm, respectively. Essential oils of lemongrass, mint and eucalyptus were inhibitory at relatively higher concentrations. The Minimum inhibitory concentration (MIC) of clove oil was 31.25 ppm by broth microdilution method. Thirty one different compounds of clove oil, constituting approximately ≥99% of the oil, were identified by gas chromatography-mass spectroscopy analysis. The major components were eugenol (75.41%), E-caryophyllene (15.11%), α-humulene (3.78%) and caryophyllene oxide (1.13%). Effect of clove oil on surface morphology of F. oxysporum f. sp. lycopersici 1322 was studied by scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM observation revealed shrivelled hyphae while AFM observation showed shrunken and disrupted spores in clove oil treated samples. In pots, 5% aqueous emulsion of clove oil controlled F. oxysporum f. sp. lycopersici 1322 infection on tomato plants. This study demonstrated clove oil as potent antifungal agent that could be used as biofungicide for the control of F. oxysporum f. sp. lycopersici in both preventive and therapeutic manner. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Enzymatic Manganese(II) Oxidation by Metabolically Dormant Spores of Diverse Bacillus Species

PubMed Central

Francis, Chris A.; Tebo, Bradley M.

2002-01-01

Bacterial spores are renowned for their longevity, ubiquity, and resistance to environmental insults, but virtually nothing is known regarding whether these metabolically dormant structures impact their surrounding chemical environments. In the present study, a number of spore-forming bacteria that produce dormant spores which enzymatically oxidize soluble Mn(II) to insoluble Mn(IV) oxides were isolated from coastal marine sediments. The highly charged and reactive surfaces of biogenic metal oxides dramatically influence the oxidation and sorption of both trace metals and organics in the environment. Prior to this study, the only known Mn(II)-oxidizing sporeformer was the marine Bacillus sp. strain SG-1, an extensively studied bacterium in which Mn(II) oxidation is believed to be catalyzed by a multicopper oxidase, MnxG. Phylogenetic analysis based on 16S rRNA and mnxG sequences obtained from 15 different Mn(II)-oxidizing sporeformers (including SG-1) revealed extensive diversity within the genus Bacillus, with organisms falling into several distinct clusters and lineages. In addition, active Mn(II)-oxidizing proteins of various sizes, as observed in sodium dodecyl sulfate-polyacrylamide electrophoresis gels, were recovered from the outer layers of purified dormant spores of the isolates. These are the first active Mn(II)-oxidizing enzymes identified in spores or gram-positive bacteria. Although extremely resistant to denaturation, the activities of these enzymes were inhibited by azide and o-phenanthroline, consistent with the involvement of multicopper oxidases. Overall, these studies suggest that the commonly held view that bacterial spores are merely inactive structures in the environment should be revised. PMID:11823231

Inhibiting Inosine Hydrolase and Alanine Racemase to Enhance the Germination of Bacillus anthracis Sterne Spores: Potential Spore Decontamination Strategies

DTIC Science & Technology

2015-06-19

animal waste an~ decompositiOn DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. UNCLASSIFIED PR-15-306 Anthrax...influx of water. Ungerminated spore Germination Germinated spore Spore hydratation ~ Non-refractile spore Refractile spore • Fluorescence

Fungal Fragments as Indoor Air Biocontaminants

PubMed Central

Górny, Rafał L.; Reponen, Tiina; Willeke, Klaus; Schmechel, Detlef; Robine, Enric; Boissier, Marjorie; Grinshpun, Sergey A.

2002-01-01

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. PMID:12089037

Spore collection and elimination apparatus and method

DOEpatents

Czajkowski, Carl [South Jamesport, NY; Warren, Barbara Panessa [Port Jefferson, NY

2007-04-03

The present invention is for a spore collection apparatus and its method of use. The portable spore collection apparatus includes a suction source, a nebulizer, an ionization chamber and a filter canister. The suction source collects the spores from a surface. The spores are activated by heating whereby spore dormancy is broken. Moisture is then applied to the spores to begin germination. The spores are then exposed to alpha particles causing extinction.

Environmental microbiology as related to planetary quarantine

NASA Technical Reports Server (NTRS)

Pflug, I. J.

1974-01-01

Continued experimental work related to dry heat resistance of microorganisms. One phase of this research has been concerned with the viability and dry heat resistance of indigenous microflora associated with small soil particles. The second part of this report is an analysis of the present status of dry heat sterilization. An attempt is made to integrate results for both laboratory grown spores and spores in soil.

Nonenzymatic microorganism identification based on ribosomal RNA

NASA Astrophysics Data System (ADS)

Ives, Jeffrey T.; Pierini, Alicia M.; Stokes, Jeffrey A.; Wahlund, Thomas M.; Read, Betsy; Bechtel, James H.; Bronk, Burt V.

1999-11-01

Effective defense against biological warfare (BW) agents requires rapid, fieldable and accurate systems. For micro- organisms like bacteria and viruses, ribosomal RNA (rRNA) provides a valuable target with multiple advantages of species specificity and intrinsic target amplification. Vegetative and spore forms of bacteria contain approximately 104 copies of rRNA. Direct detection of rRNA copies can eliminate some of the interference and preparation difficulties involved in enzymatic amplification methods. In order to apply the advantages of rRNA to BW defense, we are developing a fieldable system based on 16S rRNA, physical disruption of the micro-organism, solid phase hybridization, and fluorescence detection. Our goals include species-specific identification, complete operation from raw sample to identification in 15 minutes or less, and compact, fieldable instrumentation. Initial work on this project has investigated the lysis and hybridization steps, the species-specificity of oligonucleotides probes, and the development of a novel electromagnetic method to physically disrupt the micro- organisms. Target bacteria have been Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). Continuing work includes further development of methods to rapidly disrupt the micro-organisms and release the rRNA, improved integration and processing, and extension to bacterial and mammalian viruses like MS2 and vesicular stomatitis virus.

Gene expression profiling of human alveolar macrophages infected by B. anthracis spores demonstrates TNF-α and NF-κb are key components of the innate immune response to the pathogen

PubMed Central

2009-01-01

Background Bacillus anthracis, the etiologic agent of anthrax, has recently been used as an agent of bioterrorism. The innate immune system initially appears to contain the pathogen at the site of entry. Because the human alveolar macrophage (HAM) plays a key role in lung innate immune responses, studying the HAM response to B. anthracis is important in understanding the pathogenesis of the pulmonary form of this disease. Methods In this paper, the transcriptional profile of B. anthracis spore-treated HAM was compared with that of mock-infected cells, and differentially expressed genes were identified by Affymetrix microarray analysis. A portion of the results were verified by Luminex protein analysis. Results The majority of genes modulated by spores were upregulated, and a lesser number were downregulated. The differentially expressed genes were subjected to Ingenuity Pathway analysis, the Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis, the Promoter Analysis and Interaction Network Toolset (PAINT) and Oncomine analysis. Among the upregulated genes, we identified a group of chemokine ligand, apoptosis, and, interestingly, keratin filament genes. Central hubs regulating the activated genes were TNF-α, NF-κB and their ligands/receptors. In addition to TNF-α, a broad range of cytokines was induced, and this was confirmed at the level of translation by Luminex multiplex protein analysis. PAINT analysis revealed that many of the genes affected by spores contain the binding site for c-Rel, a member of the NF-κB family of transcription factors. Other transcription regulatory elements contained in many of the upregulated genes were c-Myb, CP2, Barbie Box, E2F and CRE-BP1. However, many of the genes are poorly annotated, indicating that they represent novel functions. Four of the genes most highly regulated by spores have only previously been associated with head and neck and lung carcinomas. Conclusion The results demonstrate not only that TNF-α and NF-κb are key components of the innate immune response to the pathogen, but also that a large part of the mechanisms by which the alveolar macrophage responds to B. anthracis are still unknown as many of the genes involved are poorly annotated. PMID:19744333

Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores

DTIC Science & Technology

2014-01-01

wild-type spores but ~15-fold higher deltaTrelease values; v ) germination kinetics of wild-type spores given a ? 30 sec 140 MPa HP pulse followed by...15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average Tlag values; and ( v ) the germination of wild-type...committed spores, as it does for nutrient-committed spores (14)? ( v ) Can these HP-com- mitted spores be isolated under conditions that do not allow

Pollen and spores of terrestrial plants

USGS Publications Warehouse

Bernhardt, Christopher E.; Willard, Debra A.; Shennan, Ian; Long, Antony J.; Horton, Benjamin P.

2015-01-01

Pollen and spores are valuable tools in reconstructing past sea level and climate because of their ubiquity, abundance, and durability as well as their reciprocity with source vegetation to environmental change (Cronin, 1999; Traverse, 2007; Willard and Bernhardt, 2011). Pollan is found in many sedimentary environments, from freshwater to saltwater, terrestrial to marine. It can be abundant in a minimal amount of sample material, for example half a gram, as concentrations can be as high as four million grains per gram (Traverse, 2007). The abundance of pollen in a sample lends it to robust statistical analysis for the quantitative reconstruction of environments. The outer cell wall is resistant to decay in sediments and allows palynomorphs (pollen and spores) to record changes in plant communities and sea level over millions of years. These characteristics make pollen and spores a powerful tool to use in sea-level research.This chapter describes the biology of pollen and spores and how they are transported and preserved in sediments. We present a methodology for isolating pollen from sediments and a general language and framework to identify pollen as well as light micrographs of a selection of common pollen grains, We then discuss their utility in sea-level research.

Reagent-free and portable detection of Bacillus anthracis spores using a microfluidic incubator and smartphone microscope.

PubMed

Hutchison, Janine R; Erikson, Rebecca L; Sheen, Allison M; Ozanich, Richard M; Kelly, Ryan T

2015-09-21

Bacillus anthracis is the causative agent of anthrax and can be contracted by humans and herbivorous mammals by inhalation, ingestion, or cutaneous exposure to bacterial spores. Due to its stability and disease potential, B. anthracis is a recognized biothreat agent and robust detection and viability methods are needed to identify spores from unknown samples. Here we report the use of smartphone-based microscopy (SPM) in combination with a simple microfluidic incubation device (MID) to detect 50 to 5000 B. anthracis Sterne spores in 3 to 5 hours. This technique relies on optical monitoring of the conversion of the ∼1 μm spores to the filamentous vegetative cells that range from tens to hundreds of micrometers in length. This distinguishing filament formation is unique to B. anthracis as compared to other members of the Bacillus cereus group. A unique feature of this approach is that the sample integrity is maintained, and the vegetative biomass can be removed from the chip for secondary molecular analysis such as PCR. Compared with existing chip-based and rapid viability PCR methods, this new approach reduces assay time by almost half, and is highly sensitive, specific, and cost effective.

The effects of salinity and temperature shock on Kappaphycus alvarezii seaweed spores release

NASA Astrophysics Data System (ADS)

Harwinda, F. K.; Satyantini, W. H.; Masithah, E. W.

2018-04-01

One of the reproductive aspects of development step that is considered as the solution of this issue is seaweed sporulation technique through which is induced through salinity and temperature shock. This study aims to determine the effect of combination and interaction of salinity and temperature shock on the release of K. alvarezii spores in order to produce superior seeds. This research was conducted using Complete Randomized Design Factorial which consists of nine combinations of treatments and three replications. The used treatment in this study is the combination of different environmental factors such as salinity shock and temperature shock. The data were analyzed using ANOVA (Analysis of Variance) followed by Duncan Multiple Range Test. The results showed that salinity (31 ppt, 33 ppt, and 35 ppt) and temperature (30°C, 32°C, and 34°C). shock affected the osmoregulation system and the release of K. alvarezii spores. The salinity shock and temperature shock had interaction with K. alvarezii spore release on the sixth and seventh day with the best treatment at 32°C temperature and 31 ppt salinity and released 5413 cells/ml spores on the seventh day.

Arbuscular mycorrhizal fungi spore propagation using single spore as starter inoculum and a plant host.

PubMed

Selvakumar, G; Shagol, C C; Kang, Y; Chung, B N; Han, S G; Sa, T M

2018-06-01

The propagation of pure cultures of arbuscular mycorrhizal fungal (AMF) is an essential requirement for their large-scale agricultural application and commercialization as biofertilizers. The present study aimed to propagate AMF using the single-spore inoculation technique and compare their propagation ability with the known reference spores. Arbuscular mycorrhizal fungal spores were collected from salt-affected Saemangeum reclaimed soil in South Korea. The technique involved inoculation of sorghum-sudangrass (Sorghum bicolor L.) seedlings with single, healthy spores on filter paper followed by the transfer of successfully colonized seedlings to 1-kg capacity pots containing sterilized soil. After the first plant cycle, the contents were transferred to 2·5-kg capacity pots containing sterilized soil. Among the 150 inoculated seedlings, only 27 seedlings were colonized by AMF spores. After 240 days, among the 27 seedlings, five inoculants resulted in the production of over 500 spores. The 18S rDNA sequencing of spores revealed that the spores produced through single-spore inoculation method belonged to Gigaspora margarita, Claroideoglomus lamellosum and Funneliformis mosseae. Furthermore, indigenous spore F. mosseae M-1 reported a higher spore count than the reference spores. The AMF spores produced using the single-spore inoculation technique may serve as potential bio-inoculants with an advantage of being more readily adopted by farmers due to the lack of requirement of a skilled technique in spore propagation. The results of the current study describe the feasible and cost-effective method to mass produce AMF spores for large-scale application. The AMF spores obtained from this method can effectively colonize plant roots and may be easily introduced to the new environment. © 2018 The Society for Applied Microbiology.

Impact of infrared treatment on quality and fungal decontamination of mung bean (Vigna radiata L.) inoculated with Aspergillus spp.

PubMed

Meenu, Maninder; Guha, Paramita; Mishra, Sunita

2018-05-01

Mung bean is a rich source of protein, carbohydrates and fiber content. It also exhibits a high level of antioxidant activity due to the presence of phenolic compounds. Aspergillus flavus and A. niger are the two major fungal strains associated with stored mung bean that lead to post-harvest losses of grains and also cause serious health risks to human beings. Thus there is a need to explore an economical decontamination method that can be used without affecting the biochemical parameters of grains. It was observed that infrared (IR) treatment of mung bean surface up to 70 °C for 5 min at an intensity of 0.299 kW m -2 led to complete visible inhibition of fungal growth. Scanning electron microscopy revealed that surface irregularities and physical disruption of spores coat are the major reasons behind the inactivation of IR-treated fungal spores. It was also reported that IR treatment up to 70 °C for 5 min does not cause any negative impact on the biochemical and physical properties of mung bean. From the results of the present study, it was concluded that IR treatment at 70 °C for 5 min using an IR source having an intensity of 0.299 kW m -2 can be successfully used as a method of fungal decontamination. The fungal spore population was reduced (approximately 5.3 log 10 CFU g -1 reductions) without significantly altering the biochemical and physical properties of grains. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Palynologic and petrographic intervals in the upper Pennsylvanian McLeansboro Group, Western Kentucky

USGS Publications Warehouse

Hower, J.C.; Helfrich, C.T.; Williams, D.A.

1994-01-01

The McLeansboro Group (formerly the Sturgis Formation) in the Western Kentucky coal field spans the upper Desmoinesian (Westphalian D) and the Missourian and Virgilian series (Stephanian). Extensive drilling has demonstrated the lateral continuity of major and minor beds in the group, making it possible to study vertical and lateral changes in palynology and petrology. Significant features of the McLeansboro Group are the marine zones over most of the coals and paleochannels, which are the only disruptions to the continuity of other lithologies. The Desmoinesian Baker (No. 13) and Wheatcroft (No. 13a) coal beds were included in the study but the primary emphasis is on the Missourian and Virgilian coals. Patoka Formation (lower Missourian) coals are dominated by tree fern spores with lesser amounts of sphenopsids, ferns and cordaites spores. This is in marked contrast to the arborescent lycopod-dominated Desmoinesian coals. Only the No. 15 coal bed exceeds 80% vitrinite: the vitrinite content of the No. 16 coal bed is less than 72%, the lowest of any Western Kentucky humic coal. The Bond Formation (upper Missourian) represents a distinct floristic interval, with a greater diversity of plant groups than in the Patoka Formation. Herbaceous lycopod spores, which are relatively minor contributors to the Patoka coals, are common in the Bond Formation. The coals generally exceed 80% vitrinite. The Mattoon Formation (Virgillian) coals have a variety of palynomorph assemblages. The low-sulfur Geiger Lake coal bed is dominated by tree fern spores, with important contributions from other ferns and sphenopsids. Similar to the underlying tree fern interval, vitrinite contents are less than 80%. The uppermost Mattoon coals are dominated by tree ferns and are notable in being the only coals more than 1 m thick in the Stephanian portion of the section, with the top coal being 4.3 m thick. The uppermost coals generally contain more than 80% vitrinite. The Permian Mauzy Formation overlies the McLeansboro Group. The palynologic/petrographic intervals appear to represent fluctuating dry (low vitrinite) and wet intervals within the Stephanian, which was itself drier than the Westphalian D. ?? 1994.

Robust Phagocyte Recruitment Controls the Opportunistic Fungal Pathogen Mucor circinelloides in Innate Granulomas In Vivo

PubMed Central

2018-01-01

ABSTRACT Mucormycosis is an emerging fungal infection with extremely high mortality rates in patients with defects in their innate immune response, specifically in functions mediated through phagocytes. However, we currently have a limited understanding of the molecular and cellular interactions between these innate immune effectors and mucormycete spores during the early immune response. Here, the early events of innate immune recruitment in response to infection by Mucor circinelloides spores are modeled by a combined in silico modeling approach and real-time in vivo microscopy. Phagocytes are rapidly recruited to the site of infection in a zebrafish larval model of mucormycosis. This robust early recruitment protects from disease onset in vivo. In silico analysis identified that protection is dependent on the number of phagocytes at the infection site, but not the speed of recruitment. The mathematical model highlights the role of proinflammatory signals for phagocyte recruitment and the importance of inhibition of spore germination for protection from active fungal disease. These in silico data are supported by an in vivo lack of fungal spore killing and lack of reactive oxygen burst, which together result in latent fungal infection. During this latent stage of infection, spores are controlled in innate granulomas in vivo. Disease can be reactivated by immunosuppression. Together, these data represent the first in vivo real-time analysis of innate granuloma formation during the early stages of a fungal infection. The results highlight a potential latent stage during mucormycosis that should urgently be considered for clinical management of patients. PMID:29588406

Characterization of fungal spores in ambient particulate matter: A study from the Himalayan region

NASA Astrophysics Data System (ADS)

Kumar, Ajay; Attri, Arun K.

2016-10-01

Fungal spores as a constituent of ambient particulate matter (PM) is of concern; they not only display the physical traits of a particle, but are also potential allergens and health risk. An investigation over fourteen month was undertaken at a rural site located in the Western Himalayan region, to evaluate the PM associated fungal spores' concentration and diversity. The season-wise change in the fungal spores concentration in the Coarse Particulate Matter (CPM) fraction (aerodynamic diameter > 10 μm) varied from 500 to 3899 spores m-3. Their average concentration over 14 months was 1517 spores m-3. Significant diversity of fungal spores in the CPM samples was observed; 27 individual genera of fungal spores were identified, of which many were known allergens. Presence of Ascomycota and Basidiomycota fungal spores was dominant in the samples; ∼20% of the spores were un-characterized. The season-wise variability in fungal spores showed a statistically significant high correlation with CPM load. Maximum number concentration of the spores in CPM was recorded in the summer, while minimum in the winter. The high diversity of spores occurred during monsoon and post monsoon months. The meteorological factors played an important role in the fungal spores' distribution profile. The temporal profile of the spores showed significant correlation with the ambient temperature (T), relative humidity (RH), wind speed (WS) and planetary boundary layer (PBL) height. Strong correlation of WS with fungal spores and CPM, and wind back trajectories suggest that re-suspension and wind assisted transport of PM contributes to ambient CPM associated fungal spores.

Viability and infectivity of fresh and cryopreserved Nosema ceranae spores.

PubMed

McGowan, Janine; De la Mora, Alvaro; Goodwin, Paul H; Habash, Marc; Hamiduzzaman, Mollah Md; Kelly, Paul G; Guzman-Novoa, Ernesto

2016-12-01

The microsporidium fungus Nosema ceranae is an intracellular parasite that infects the midgut of the honey bee, Apis mellifera. A major limitation of research on N. ceranae is that the fungus is non-culturable and thus studying it depends on the seasonal availability of Nosema spores. Also, spore viability and infectivity can vary considerably, and thus there is a need for reliable methods for determining those traits. This study examined different conditions for N. ceranae spore cryopreservation at -70°C, assessing spore viability and infectivity. Viability was determined by a staining procedure counting total spores numbers with bright field microscopy and un-viable spore numbers with the fluorescent dye, propidium iodide. Spore infectivity was determined with a dilution inoculation assay. Infectivity was dependent on the inoculum dose for the proportion of bees with detectable Nosema infections based on the number of spores per bee at 18days after inoculation; 4000 spores per bee or higher were needed to get approx. 100% of the inoculated bees infected. The median infective dose (ID 50 ) was 149 spores per bee, and the minimum dose capable of causing a detectable infection was 1.28 spores. The proportion of N. ceranae infected bees correlated significantly with the number of spores per bee (r=0.98, P<0.0001). N. ceranae spores cryopreserved in water or 10% glycerol did not differ in viability compared to fresh spores, but lost infectivity when inoculated into bees. This study shows that while cryopreservation of N. ceranae spores can preserve viability, the spores can have reduced infectivity. Copyright © 2016 Elsevier B.V. All rights reserved.

The Bacillus subtilis yabG Gene Is Transcribed by SigK RNA Polymerase during Sporulation, and yabG Mutant Spores Have Altered Coat Protein Composition

PubMed Central

Takamatsu, Hiromu; Kodama, Takeko; Imamura, Atsuo; Asai, Kei; Kobayashi, Kazuo; Nakayama, Tatsuo; Ogasawara, Naotake; Watabe, Kazuhito

2000-01-01

The expression of six novel genes located in the region from abrB to spoVC of the Bacillus subtilis chromosome was analyzed, and one of the genes, yabG, had a predicted promoter sequence conserved among SigK-dependent genes. Northern blot analysis revealed that yabG mRNA was first detected from 4 h after the cessation of logarithmic growth (T4) in wild-type cells and in a gerE36 (GerE−) mutant but not in spoIIAC (SigF−), spoIIGAB (SigE−), spoIIIG (SigG−), and spoIVCB (SigK−) mutants. The transcription start point was determined by primer extension analysis; the −10 and −35 regions are very similar to the consensus sequences recognized by SigK-containing RNA polymerase. Inactivation of the yabG gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yabG spores in l-alanine and in a mixture of l-asparagine, d-glucose, d-fructose, and potassium chloride was also the same as that of wild-type spores. On the other hand, the protein preparation from yabG spores included 15-, 18-, 21-, 23-, 31-, 45-, and 55-kDa polypeptides which were low in or not extracted from wild-type spores under the same conditions. We determined their N-terminal amino acid sequence and found that these polypeptides were CotT, YeeK, YxeE, CotF, YrbA (31 and 45 kDa), and SpoIVA, respectively. The fluorescence of YabG-green fluorescent protein fusion produced in sporulating cells was detectable in the forespores but not in the mother cell compartment under fluorescence microscopy. These results indicate that yabG encodes a sporulation-specific protein which is involved in coat protein composition in B. subtilis. PMID:10714992

Temporal regulation and forespore-specific expression of the spore photoproduct lyase gene by sigma-G RNA polymerase during Bacillus subtilis sporulation.

PubMed Central

Pedraza-Reyes, M; Gutiérrez-Corona, F; Nicholson, W L

1994-01-01

Bacterial spores are highly resistant to killing by UV radiation and exhibit unique DNA photochemistry. UV irradiation of spore DNA results in formation of spore photoproduct (SP), the thymine dimer 5-thyminyl-5,6-dihydrothymine. Repair of SP occurs during germination of Bacillus subtilis spores by two distinct routes, either by the general nucleotide excision repair (uvr) pathway or by a novel SP-specific monomerization reaction mediated by the enzyme SP lyase, which is encoded by the spl gene. Repair of SP occurs early in spore germination and is independent of de novo protein synthesis, suggesting that the SP repair enzymes are synthesized during sporulation and are packaged in the dormant spore. To test this hypothesis, the expression of a translational spl-lacZ fusion integrated at the spl locus was monitored during B. subtilis growth and sporulation. beta-Galactosidase expression from the spl-lacZ fusion was silent during vegetative growth and was not DNA damage inducible, but it was activated at morphological stage III of sporulation specifically in the forespore compartment, coincident with activation of expression of the stage III marker enzyme glucose dehydrogenase. Expression of the spl-lacZ fusion was shown to be dependent upon the sporulation-specific RNA polymerase containing the sigma-G factor (E sigma G), as spl-lacZ expression was abolished in a mutant harboring a deletion in the sigG gene and restored by expression of the sigG gene in trans. Primer extension analysis of spl mRNA revealed a major extension product initiating upstream from a small open reading frame of unknown function which precedes spl, and it revealed two other shorter minor extension products. All three extension products were present in higher quantities during sporulation and after sigG induction. The three putative transcripts are all preceded by sequences which share homology with the consensus sigma-G factor-type promoter sequence, but in vitro transcription by purified sigma-G RNA polymerase was detected only from the promoter corresponding to the major extension product. The open reading frame-spl operon therefore appears to be an additional member of the sigma-G regulon, which also includes as members the small, acid-soluble spore proteins which are in large part responsible for spore DNA photochemistry. Therefore, sporulating bacteria appear to coordinately regulate genes whose products not only alter spore DNA photochemistry but also repair the major spore-specific photoproduct during germination Images PMID:8021181

Myxidium scripta n. sp. identified in urinary and biliary tract of Louisiana-farmed red-eared slider turtles Trachemys scripta elegans.

PubMed

Roberts, John F; Whipps, Christopher M; Bartholomew, Jerri L; Schneider, Lynda; Jacobson, Elliott R

2008-08-07

During a necropsy investigation of a mortality event occurring at a turtle farm in Assumption Parish, Louisiana, spores of a myxozoan were identified in the renal tubules in 3 of 6, the gall bladder lumen in 2 of 6, and the bile ductule in 1 of 6 red eared slider turtles Trachemys scripta elegans. In total, myxozoa were identified in 4 of 6 turtles. In 1 turtle, renal tubules contained numerous mature spores, had epithelial hyperplasia, granulomatous transformation, compression of adjacent tubules and interstitial lymphocytic nephritis. The genus of myxozoan was Myxidium, based on spore morphology in cytological preparations, in histologic section, and by electron microscopy. In cytological preparation the spores had mean dimensions of 18.8 x 5.1 microm and a mean polar capsule dimension of 6.6 x 3.5 microm. Electron microscopy showed renal tubules contained plasmodia with disporoblasts with spores in various stages of maturation. Ultrastructure of mature spores demonstrated a capsule containing 2 asymmetrical overlapping valves and polar capsules containing a polar filament coiled 6 to 8 times and surrounded by a membrane composed of a double layer wall. The small subunit rDNA gene sequence was distinct from all other Myxidium species for which sequences are available. Additionally, this is the first Myxidium species recovered from a North American chelonian to receive genetic analysis. Although T. s. elegans is listed as a host for Myxidium chelonarum, this newly described species of Myxidium possessed larger spores with tapered ends; thus, we described it as a new species, Myxidium scripta n. sp. This report documents a clinically significant nephropathy and genetic sequence from a Myxidium parasite affecting a freshwater turtle species in North America.

Integration of Membrane Distillation with solar photo-Fenton for purification of water contaminated with Bacillus sp. and Clostridium sp. spores.

PubMed

Ruiz-Aguirre, A; Polo-López, M I; Fernández-Ibáñez, P; Zaragoza, G

2017-10-01

Although Membrane Distillation (MD) has been extensively studied for desalination, it has other applications like removing all kinds of solutes from water and concentrating non-volatile substances. MD offers the possibility of producing a clean stream while concentrating valuable compounds from waste streams towards their recovery, or emerging contaminants and pathogens present in wastewater in order to facilitate their chemical elimination. This paper analyses the elimination of bacterial spores from contaminated water with MD and the role of MD in the subsequent treatment of the concentrate with photo-Fenton process. The experiments were performed at Plataforma Solar de Almería (PSA) using a plate and frame bench module with a Permeate Gap Membrane Distillation (PGMD) configuration. Tests were done for two different kinds of spores in two different water matrixes: distilled water with 3.5wt% of sea salts contaminated with spores of Bacillus subtilis (B. subtilis) and wastewater after a secondary treatment and still contaminated with Clostridium sp. spores. An analysis of the permeate was performed in all cases to determine its purity, as well as the concentrated stream and its further treatment in order to assess the benefits of using MD. Results showed a permeate free of spores in all the cases, demonstrating the viability of MD to treat biological contaminated wastewater for further use in agriculture. Moreover, the results obtained after treating the concentrate with photo-Fenton showed a shorter treatment time for the reduction of the spore concentration in the water than that when only photo-Fenton was used. Copyright © 2017 Elsevier B.V. All rights reserved.

Biodiversity of arbuscular mycorrhizal fungi in roots and soils of two salt marshes.

PubMed

Wilde, Petra; Manal, Astrid; Stodden, Marc; Sieverding, Ewald; Hildebrandt, Ulrich; Bothe, Hermann

2009-06-01

The occurrence of arbuscular mycorrhizal fungi (AMF) was assessed by both morphological and molecular criteria in two salt marshes: (i) a NaCl site of the island Terschelling, Atlantic Coast, the Netherlands and (ii) a K(2)CO(3) marsh at Schreyahn, Northern Germany. The overall biodiversity of AMF, based on sequence analysis, was comparably low in roots at both sites. However, the morphological spore analyses from soil samples of both sites exhibited a higher AMF biodiversity. Glomus geosporum was the only fungus of the Glomerales that was detected both as spores in soil samples and in roots of the AMF-colonized salt plants Aster tripolium and Puccinellia sp. at both saline sites and on all sampling dates (one exception). In roots, sequences of Glomus intraradices prevailed, but this fungus could not be identified unambiguously from DNA of soil spores. Likewise, Glomus sp. uncultured, only deposited as sequence in the database, was widely detected by DNA sequencing in root samples. All attempts to obtain the corresponding sequences from spores isolated from soil samples failed consistently. A small sized Archaeospora sp. was detected, either/or by morphological and molecular analyses, in roots or soil spores, in dead AMF spores or orobatid mites. The study noted inconsistencies between morphological characterization and identification by DNA sequencing of the 5.8S rDNA-ITS2 region or part of the 18S rDNA gene. The distribution of AMF unlikely followed the salt gradient at both sites, in contrast to the zone formation of plant species. Zygotes of the alga Vaucheria erythrospora (Xanthophyceae) were retrieved and should not be misidentified with AMF spores.

Culturability of Bacillus spores on aerosol collection filters exposed to airborne combustion products of Al, Mg, and B·Ti.

PubMed

Adhikari, Atin; Yermakov, Michael; Indugula, Reshmi; Reponen, Tiina; Driks, Adam; Grinshpun, Sergey A

2016-05-01

Destruction of bioweapon facilities due to explosion or fire could aerosolize highly pathogenic microorganisms. The post-event air quality assessment is conducted through air sampling. A bioaerosol sample (often collected on a filter for further culture-based analysis) also contains combustion products, which may influence the microbial culturability and, thus, impact the outcome. We have examined the interaction between spores deposited on collection filters using two simulants of Bacillus anthracis [B. thuringiensis (Bt) and B. atrophaeus (referred to as BG)] and incoming combustion products of Al as well as Mg and B·Ti (common ingredient of metalized explosives). Spores extracted from Teflon, polycarbonate, mixed cellulose ester (MCE), and gelatin filters (most common filter media for bioaerosol sampling), which were exposed to combustion products during a short-term sampling, were analyzed by cultivation. Surprisingly, we observed that aluminum combustion products enhanced the culturability of Bt (but not BG) spores on Teflon filters increasing the culturable count by more than an order of magnitude. Testing polycarbonate and MCE filter materials also revealed a moderate increase of culturability although gelatin did not. No effect was observed with either of the two species interacting on either filter media with products originated by combustion of Mg and B·Ti. Sample contamination, spore agglomeration, effect of a filter material on the spore survival, changes in the spore wall ultrastructure and germination, as well as other factors were explored to interpret the findings. The study raises a question about the reliability of certain filter materials for collecting airborne bio-threat agents in combustion environments. Copyright © 2016 Elsevier Inc. All rights reserved.

Selection of inactivation medium for fungal spores in clinical wastes by supercritical carbon dioxide.

PubMed

Noman, Efaq; Norulaini Nik Ab Rahman, Nik; Al-Gheethi, Adel; Nagao, Hideyuki; Talip, Balkis A; Ab Kadir, Omar

2018-05-21

The present study aimed to select the best medium for inactivation of Aspergillus fumigatus, Aspergillus spp. in section Nigri, A. niger, A. terreus var. terreus, A. tubingensis, Penicillium waksmanii, P. simplicissimum, and Aspergillus sp. strain no. 145 spores in clinical wastes by using supercritical carbon dioxide (SC-CO 2 ). There were three types of solutions used including normal saline, seawater, distilled water, and physiological saline with 1% of methanol; each solution was tested at 5, 10, and 20 mL of the water contents. The experiments were conducted at the optimum operating parameters of supercritical carbon dioxide (30 MPa, 75 °C, 90 min). The results showed that the inactivation rate was more effective in distilled water with the presence of 1% methanol (6 log reductions). Meanwhile, the seawater decreases inactivation rate more than normal saline (4.5 vs. 5.1 log reduction). On the other hand, the experiments performed with different volumes of distilled water (5, 10, and 20 mL) indicated that A. niger spores were completely inactivated with 10 mL of distilled water. The inactivation rate of fungal spores decreased from 6 to 4.5 log as the amount of distilled water increased from 10 to 20 mL. The analysis for the spore morphology of A. fumigatus and Aspergillus spp. in section Nigri using scanning electron microscopy (SEM) has revealed the role of temperature and pressure in the SC-CO 2 in the destruction of the cell walls of the spores. It can be concluded that the distilled water represent the best medium for inactivation of fungal spores in the clinical solid wastes by SC-CO 2 .

Effect of Nanoencapsulated Vitamin B1 Derivative on Inhibition of Both Mycelial Growth and Spore Germination of Fusarium oxysporum f. sp. raphani

PubMed Central

Cho, Jeong Sub; Seo, Yong Chang; Yim, Tae Bin; Lee, Hyeon Yong

2013-01-01

Nanoencapsulation of thiamine dilauryl sulfate (TDS), a vitamin B1 derivative, was proved to effectively inhibit the spore germination of Fusarium oxysporum f. sp. raphani (F. oxysporum), as well as mycelial growth. The average diameter of nanoparticles was measured as 136 nm by being encapsulated with an edible encapsulant, lecithin, whose encapsulation efficiency was about 55% in containing 200 ppm of TDS concentration: the 100 ppm TDS nanoparticle solution showed a mycelial growth inhibition rate of 59%. These results were about similar or even better than the cases of treating 100 ppm of dazomet, a positive antifungal control (64%). Moreover, kinetic analysis of inhibiting spore germination were estimated as 6.6% reduction of spore germination rates after 24 h treatment, which were 3.3% similar to the case of treating 100 ppm of a positive control (dazomet) for the same treatment time. It was also found that TDS itself could work as an antifungal agent by inhibiting both mycelial growth and spore germination, even though its efficacy was lower than those of nanoparticles. Nanoparticles especially played a more efficient role in limiting the spore germination, due to their easy penetration into hard cell membranes and long resident time on the surface of the spore shell walls. In this work, it was first demonstrated that the nanoparticle of TDS not a harmful chemical can control the growth of F. oxysporum by using a lower dosage than commercial herbicides, as well as the inhibiting mechanism of the TDS. However, field trials of the TDS nanoparticles encapsulated with lecithin should be further studied to be effectively used for field applications. PMID:23429270

REVERSIBLE ACTIVATION FOR GERMINATION AND SUBSEQUENT CHANGES IN BACTERIAL SPORES1

PubMed Central

Lee, W. H.; Ordal, Z. John

1963-01-01

Lee, W. H. (University of Illinois, Urbana) and Z. John Ordal. Reversible activation for germination and subsequent changes in bacterial spores. J. Bacteriol. 85:207–217. 1963.—It was possible to isolate refractile spores of Bacillus megaterium, from a calcium dipicolinate germination solution, that were activated and would germinate spontaneously in distilled water. Some of the characteristics of the initial phases of bacterial spore germination were determined by studying these unstable activated spores. Activated spores of B. megaterium were resistant to stains and possessed a heat resistance intermediate between that of dormant and of germinated spores. The spontaneous germination of activated spores was inhibited by copper, iron, silver, or mercury salts, saturated o-phenanthroline, or solutions having a low pH value, but not by many common inhibitors. These inhibitions could be partially or completely reversed by the addition of sodium dipicolinate. The activated spores could be deactivated and made similar to dormant spores by treatment with acid. Analyses of the exudates from the variously treated spore suspensions revealed that whatever inhibited the germination of activated spores also inhibited the release of spore material. The composition of the germination exudates was different than that of extracts of dormant spores. Although heavy suspensions of activated spores gradually became swollen and dark when suspended in solutions of o-phenanthroline or at pH 4, the materials released resembled those found in extracts of dormant spores rather than those of normal germination exudates. Images PMID:16561987

A new species of myxozoan (Myxosporea) from the brain and spinal cord of rainbow trout (Oncorhynchus mykiss) from Idaho.

PubMed

Hogge, Carla I; Campbell, Matthew R; Johnson, Keith A

2008-02-01

A new species of Myxosporea, Myxobolus neurotropus n. sp., is described from the brain and spinal cord of rainbow trout (Oncorhynchus mykiss) from Duncan Creek, Owyhee County, Idaho. Spores are oval, have 2 pyriform polar capsules, and possess a thick spore wall (sutural rim) with a short intracapsular offshoot. The mean spore dimensions are length 11.8 microm, width 10.8 microm, and thickness 8.8 microm. This myxozoan is compared to other described Myxobolus species found in cranial tissues of salmonids in terms of spore morphology and phylogenetic analysis. Because it is found in brain and spinal cord, it is encountered while performing screening tests for Myxobolus cerebralis, the causative agent of salmonid whirling disease. Where chronic inflammation and granulomatous lesions are associated with M. cerebralis, histological examination shows no host response to M. neurotropus n. sp. A diagnostic polymerase chain reaction (PCR) test is included as an aid in properly identifying the species.

The differential effects of heat-shocking on the viability of spores from Bacillus anthracis, Bacillus subtilis, and Clostridium sporogenes after treatment with peracetic acid- and glutaraldehyde-based disinfectants

PubMed Central

March, Jordon K; Pratt, Michael D; Lowe, Chinn-Woan; Cohen, Marissa N; Satterfield, Benjamin A; Schaalje, Bruce; O'Neill, Kim L; Robison, Richard A

2015-01-01

This study investigated (1) the susceptibility of Bacillus anthracis (Ames strain), Bacillus subtilis (ATCC 19659), and Clostridium sporogenes (ATCC 3584) spores to commercially available peracetic acid (PAA)- and glutaraldehyde (GA)-based disinfectants, (2) the effects that heat-shocking spores after treatment with these disinfectants has on spore recovery, and (3) the timing of heat-shocking after disinfectant treatment that promotes the optimal recovery of spores deposited on carriers. Suspension tests were used to obtain inactivation kinetics for the disinfectants against three spore types. The effects of heat-shocking spores after disinfectant treatment were also determined. Generalized linear mixed models were used to estimate 6-log reduction times for each spore type, disinfectant, and heat treatment combination. Reduction times were compared statistically using the delta method. Carrier tests were performed according to AOAC Official Method 966.04 and a modified version that employed immediate heat-shocking after disinfectant treatment. Carrier test results were analyzed using Fisher's exact test. PAA-based disinfectants had significantly shorter 6-log reduction times than the GA-based disinfectant. Heat-shocking B. anthracis spores after PAA treatment resulted in significantly shorter 6-log reduction times. Conversely, heat-shocking B. subtilis spores after PAA treatment resulted in significantly longer 6-log reduction times. Significant interactions were also observed between spore type, disinfectant, and heat treatment combinations. Immediately heat-shocking spore carriers after disinfectant treatment produced greater spore recovery. Sporicidal activities of disinfectants were not consistent across spore species. The effects of heat-shocking spores after disinfectant treatment were dependent on both disinfectant and spore species. Caution must be used when extrapolating sporicidal data of disinfectants from one spore species to another. Heat-shocking provides a more accurate picture of spore survival for only some disinfectant/spore combinations. Collaborative studies should be conducted to further examine a revision of AOAC Official Method 966.04 relative to heat-shocking. PMID:26185111

Multifactorial Resistance of Bacillus subtilis Spores to High-Energy Proton Radiation: Role of Spore Structural Components and the Homologous Recombination and Non-Homologous End Joining DNA Repair Pathways

PubMed Central

Reitz, Günther; Li, Zuofeng; Klein, Stuart; Nicholson, Wayne L.

2012-01-01

Abstract The space environment contains high-energy charged particles (e.g., protons, neutrons, electrons, α-particles, heavy ions) emitted by the Sun and galactic sources or trapped in the radiation belts. Protons constitute the majority (87%) of high-energy charged particles. Spores of Bacillus species are one of the model systems used for astro- and radiobiological studies. In this study, spores of different Bacillus subtilis strains were used to study the effects of high energetic proton irradiation on spore survival. Spores of the wild-type B. subtilis strain [mutants deficient in the homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways and mutants deficient in various spore structural components such as dipicolinic acid (DPA), α/β-type small, acid-soluble spore protein (SASP) formation, spore coats, pigmentation, or spore core water content] were irradiated as air-dried multilayers on spacecraft-qualified aluminum coupons with 218 MeV protons [with a linear energy transfer (LET) of 0.4 keV/μm] to various final doses up to 2500 Gy. Spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to proton radiation than wild-type spores, indicating that both HR and NHEJ DNA repair pathways are needed for spore survival. Spores lacking DPA, α/β-type SASP, or with increased core water content were also significantly more sensitive to proton radiation, whereas the resistance of spores lacking pigmentation or spore coats was essentially identical to that of the wild-type spores. Our results indicate that α/β-type SASP, core water content, and DPA play an important role in spore resistance to high-energy proton irradiation, suggesting their essential function as radioprotectants of the spore interior. Key Words: Bacillus—Spores—DNA repair—Protection—High-energy proton radiation. Astrobiology 12, 1069–1077. PMID:23088412

Distribution of Paenibacillus larvae spores inside honey bee colonies and its relevance for diagnosis.

PubMed

Gillard, M; Charriere, J D; Belloy, L

2008-09-01

One of the most important factors affecting the development of honey bee colonies is infectious diseases such as American foulbrood (AFB) caused by the spore forming Gram-positive bacterium Paenibacillus larvae. Colony inspections for AFB clinical symptoms are time consuming. Moreover, diseased cells in the early stages of the infection may easily be overlooked. In this study, we investigated whether it is possible to determine the sanitary status of a colony based on analyses of different materials collected from the hive. We analysed 237 bee samples and 67 honey samples originating from 71 colonies situated in 13 apiaries with clinical AFB occurrences. We tested whether a difference in spore load among bees inside the whole hive exists and which sample material related to its location inside the hive was the most appropriate for an early AFB diagnosis based on the culture method. Results indicated that diagnostics based on analysis of honey samples and bees collected at the hive entrance are of limited value as only 86% and 83%, respectively, of samples from AFB-symptomatic colonies were positive. Analysis of bee samples collected from the brood nest, honey chamber, and edge frame allowed the detection of all colonies showing AFB clinical symptoms. Microbiological analysis showed that more than one quarter of samples collected from colonies without AFB clinical symptoms were positive for P. larvae. Based on these results, we recommend investigating colonies by testing bee samples from the brood nest, edge frame or honey chamber for P. larvae spores.

Sporulation environment influences spore properties in Bacillus: evidence and insights on underlying molecular and physiological mechanisms.

PubMed

Bressuire-Isoard, Christelle; Broussolle, Véronique; Carlin, Frédéric

2018-05-17

Bacterial spores are resistant to physical and chemical insults, which make them a major concern for public health and for industry. Spores help bacteria to survive extreme environmental conditions that vegetative cells cannot tolerate. Spore resistance and dormancy are important properties for applications in medicine, veterinary health, food safety, crop protection, and other domains. The resistance of bacterial spores results from a protective multilayered structure and from the unique composition of the spore core. The mechanisms of sporulation and germination, the first stage after breaking of dormancy, and organization of spore structure have been extensively studied in Bacillus species. This review aims to illustrate how far the structure, composition and properties of spores are shaped by the environmental conditions in which spores form. We look at the physiological and molecular mechanisms underpinning how sporulation media and environment deeply affect spore yield, spore properties like resistance to wet heat and physical and chemical agents, germination, and further growth. For example, spore core water content decreases as sporulation temperature increases, and resistance to wet heat increases. Controlling the fate of Bacillus spores is pivotal to controlling bacterial risks and process efficiencies in, for example, the food industry, and better control hinges on better understanding how sporulation conditions influence spore properties.

Bacillus anthracis spore movement does not require a carrier cell and is not affected by lethal toxin in human lung models.

PubMed

Booth, J Leland; Duggan, Elizabeth S; Patel, Vineet I; Langer, Marybeth; Wu, Wenxin; Braun, Armin; Coggeshall, K Mark; Metcalf, Jordan P

2016-10-01

The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores escape from the alveolus to regional lymph nodes, germinate and enter the circulatory system to cause disease. The roles of carrier cells and the effects of B. anthracis toxins in this process are unclear. We used a human lung organ culture model to measure spore uptake by antigen presenting cells (APC) and alveolar epithelial cells (AEC), spore partitioning between these cells, and the effects of B. anthracis lethal toxin and protective antigen. We repeated the study in a human A549 alveolar epithelial cell model. Most spores remained unassociated with cells, but the majority of cell-associated spores were in AEC, not in APC. Spore movement was not dependent on internalization, although the location of internalized spores changed in both cell types. Spores also internalized in a non-uniform pattern. Toxins affected neither transit of the spores nor the partitioning of spores into AEC and APC. Our results support a model of spore escape from the alveolus that involves spore clustering with transient passage through intact AEC. However, subsequent transport of spores by APC from the lung to the lymph nodes may occur. Published by Elsevier Masson SAS.

Thermodynamic analysis of Bacillus subtilis endospore protonation using isothermal titration calorimetry

NASA Astrophysics Data System (ADS)

Harrold, Zoë R.; Gorman-Lewis, Drew

2013-05-01

Bacterial proton and metal adsorption reactions have the capacity to affect metal speciation and transport in aqueous environments. We coupled potentiometric titration and isothermal titration calorimetry (ITC) analyses to study Bacillus subtilis spore-proton adsorption. We modeled the potentiometric data using a four and five-site non-electrostatic surface complexation model (NE-SCM). Heats of spore surface protonation from coupled ITC analyses were used to determine site specific enthalpies of protonation based on NE-SCMs. The five-site model resulted in a substantially better model fit for the heats of protonation but did not significantly improve the potentiometric titration model fit. The improvement observed in the five-site protonation heat model suggests the presence of a highly exothermic protonation reaction circa pH 7 that cannot be resolved in the less sensitive potentiometric data. From the log Ks and enthalpies we calculated corresponding site specific entropies. Log Ks and site concentrations describing spore surface protonation are statistically equivalent to B. subtilis cell surface protonation constants. Spore surface protonation enthalpies, however, are more exothermic relative to cell based adsorption suggesting a different bonding environment. The thermodynamic parameters defined in this study provide insight on molecular scale spore-surface protonation reactions. Coupled ITC and potentiometric titrations can reveal highly exothermic, and possibly endothermic, adsorption reactions that are overshadowed in potentiometric models alone. Spore-proton adsorption NE-SCMs derived in this study provide a framework for future metal adsorption studies.

Testing Nucleoside Analogues as Inhibitors of Bacillus anthracis Spore Germination In Vitro and in Macrophage Cell Culture ▿

PubMed Central

Alvarez, Zadkiel; Lee, Kyungae; Abel-Santos, Ernesto

2010-01-01

Bacillus anthracis, the etiological agent of anthrax, has a dormant stage in its life cycle known as the endospore. When conditions become favorable, spores germinate and transform into vegetative bacteria. In inhalational anthrax, the most fatal manifestation of the disease, spores enter the organism through the respiratory tract and germinate in phagosomes of alveolar macrophages. Germinated cells can then produce toxins and establish infection. Thus, germination is a crucial step for the initiation of pathogenesis. B. anthracis spore germination is activated by a wide variety of amino acids and purine nucleosides. Inosine and l-alanine are the two most potent nutrient germinants in vitro. Recent studies have shown that germination can be hindered by isomers or structural analogues of germinants. 6-Thioguanosine (6-TG), a guanosine analogue, is able to inhibit germination and prevent B. anthracis toxin-mediated necrosis in murine macrophages. In this study, we screened 46 different nucleoside analogues as activators or inhibitors of B. anthracis spore germination in vitro. These compounds were also tested for their ability to protect the macrophage cell line J774a.1 from B. anthracis cytotoxicity. Structure-activity relationship analysis of activators and inhibitors clarified the binding mechanisms of nucleosides to B. anthracis spores. In contrast, no structure-activity relationships were apparent for compounds that protected macrophages from B. anthracis-mediated killing. However, multiple inhibitors additively protected macrophages from B. anthracis. PMID:20921305

Understanding of the importance of the spore coat structure and pigmentation in the Bacillus subtilis spore resistance to low-pressure plasma sterilization

NASA Astrophysics Data System (ADS)

Raguse, Marina; Fiebrandt, Marcel; Denis, Benjamin; Stapelmann, Katharina; Eichenberger, Patrick; Driks, Adam; Eaton, Peter; Awakowicz, Peter; Moeller, Ralf

2016-07-01

Low-pressure plasmas have been evaluated for their potential in biomedical and defense purposes. The sterilizing effect of plasma can be attributed to several active agents, including (V)UV radiation, charged particles, radical species, neutral and excited atoms and molecules, and the electric field. Spores of Bacillus subtilis were used as a bioindicator and a genetic model system to study the sporicidal effects of low-pressure plasma decontamination. Wild-type spores, spores lacking the major protective coat layers (inner, outer, and crust), pigmentation-deficient spores or spore impaired in encasement (a late step in coat assembly) were systematically tested for their resistance to low-pressure argon, hydrogen, and oxygen plasmas with and without admixtures. We demonstrate that low-pressure plasma discharges of argon and oxygen discharges cause significant physical damage to spore surface structures as visualized by atomic force microscopy. Spore resistance to low-pressure plasma was primarily dependent on the presence of the inner, and outer spore coat layers as well as spore encasement, with minor or less importance of the crust and spore pigmentation, whereas spore inactivation itself was strongly influenced by the gas composition and operational settings.

Bacteria, mould and yeast spore inactivation studies by scanning electron microscope observations.

PubMed

Rozali, Siti N M; Milani, Elham A; Deed, Rebecca C; Silva, Filipa V M

2017-12-18

Spores are the most resistant form of microbial cells, thus difficult to inactivate. The pathogenic or food spoilage effects of certain spore-forming microorganisms have been the primary basis of sterilization and pasteurization processes. Thermal sterilization is the most common method to inactivate spores present on medical equipment and foods. High pressure processing (HPP) is an emerging and commercial non-thermal food pasteurization technique. Although previous studies demonstrated the effectiveness of thermal and non-thermal spore inactivation, the in-depth mechanisms of spore inactivation are as yet unclear. Live and dead forms of two food spoilage bacteria, a mould and a yeast were examined using scanning electron microscopy before and after the inactivation treatment. Alicyclobacillus acidoterrestris and Geobacillus stearothermophilus bacteria are indicators of acidic foods pasteurization and sterilization processes, respectively. Neosartorya fischeri is a phyto-pathogenic mould attacking fruits. Saccharomyces cerevisiae is a yeast with various applications for winemaking, brewing, baking and the production of biofuel from crops (e.g. sugar cane). Spores of the four microbial species were thermally inactivated. Spores of S. cerevisiae were observed in the ascus and free form after thermal and HPP treatments. Different forms of damage and cell destruction were observed for each microbial spore. Thermal treatment inactivated bacterial spores of A. acidoterrestris and G. stearothermophilus by attacking the inner core of the spore. The heat first altered the membrane permeability allowing the release of intracellular components. Subsequently, hydration of spores, physicochemical modifications of proteins, flattening and formation of indentations occurred, with subsequent spore death. Regarding N. fischeri, thermal inactivation caused cell destruction and leakage of intracellular components. Both thermal and HPP treatments of S. cerevisiae free spores attacked the inner membrane, altering its permeability, and allowing in final stages the transfer of intracellular components to the outside. The spore destruction caused by thermal treatment was more severe than HPP, as HPP had less effect on the spore core. All injured spores have undergone irreversible volume and shape changes. While some of the leakage of spore contents is visible around the deformed but fully shaped spore, other spores exhibited large indentations and were completely deformed, apparently without any contents inside. This current study contributed to the understanding of spore inactivation by thermal and non-thermal processes. Copyright © 2017 Elsevier B.V. All rights reserved.

Asynchronous spore germination in isogenic natural isolates of Saccharomyces paradoxus.

PubMed

Stelkens, Rike B; Miller, Eric L; Greig, Duncan

2016-05-01

Spores from wild yeast isolates often show great variation in the size of colonies they produce, for largely unknown reasons. Here we measure the colonies produced from single spores from six different wild Saccharomyces paradoxus strains. We found remarkable variation in spore colony sizes, even among spores that were genetically identical. Different strains had different amounts of variation in spore colony sizes, and variation was not affected by the number of preceding meioses, or by spore maturation time. We used time-lapse photography to show that wild strains also have high variation in spore germination timing, providing a likely mechanism for the variation in spore colony sizes. When some spores from a laboratory strain make small colonies, or no colonies, it usually indicates a genetic or meiotic fault. Here, we demonstrate that in wild strains spore colony size variation is normal. We discuss and assess potential adaptive and non-adaptive explanations for this variation. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Role of Visible Light-Activated Photocatalyst on the Reduction of Anthrax Spore-Induced Mortality in Mice

PubMed Central

Huang, Hsin-Hsien; Wong, Ming-Show; Lin, Hung-Chi; Chang, Hsin-Hou

2009-01-01

Background Photocatalysis of titanium dioxide (TiO2) substrates is primarily induced by ultraviolet light irradiation. Anion-doped TiO2 substrates were shown to exhibit photocatalytic activities under visible-light illumination, relative environmentally-friendly materials. Their anti-spore activity against Bacillus anthracis, however, remains to be investigated. We evaluated these visible-light activated photocatalysts on the reduction of anthrax spore-induced pathogenesis. Methodology/Principal Findings Standard plating method was used to determine the inactivation of anthrax spore by visible light-induced photocatalysis. Mouse models were further employed to investigate the suppressive effects of the photocatalysis on anthrax toxin- and spore-mediated mortality. We found that anti-spore activities of visible light illuminated nitrogen- or carbon-doped titania thin films significantly reduced viability of anthrax spores. Even though the spore-killing efficiency is only approximately 25%, our data indicate that spores from photocatalyzed groups but not untreated groups have a less survival rate after macrophage clearance. In addition, the photocatalysis could directly inactivate lethal toxin, the major virulence factor of B. anthracis. In agreement with these results, we found that the photocatalyzed spores have tenfold less potency to induce mortality in mice. These data suggest that the photocatalysis might injury the spores through inactivating spore components. Conclusion/Significance Photocatalysis induced injuries of the spores might be more important than direct killing of spores to reduce pathogenicity in the host. PMID:19132100

Optical and biological properties of plasma-treated Neurospora crassa spores as studied by absorption, circular dichroism, and Raman spectroscopy

NASA Astrophysics Data System (ADS)

Lee, Geon Joon; Park, Gyungsoon; Choi, Eun Ha

2017-11-01

We studied the effect of plasma treatment on the optical, structural and biological properties of Neurospora crassa ( N. crassa) spores. An atmospheric-pressure plasma jet (APPJ) was used to generate reactive oxygen and nitrogen species in aqueous solution. The APPJ treatment of N. crassa spores in water significantly reduced the viability of spores. The reduction in the spore viability can be attributed to the reactive species from the plasma itself and those derived from the reaction of plasma radicals with aqueous solution. These structural modifications were contingent on the medium in which N. crassa spores were suspended; plasma treatment of N. crassa spores in PBS did not significantly affect the viability of spores as compared with N. crassa spores in water. Scanning electron microscopy images and circular dichroism spectra indicated that the spore cell wall was damaged by plasma treatment. The optical absorption spectrum of untreated N. crassa spores exhibited two resonance absorption bands at approximately λ1 ≈ 260 nm and λ2 ≈ 472 nm, originating from deoxyribonucleic acid (DNA) and β-carotene. The Raman spectrum of untreated N. crassa spores exhibited three main peaks at 1519, 1157 and 1006 cm -1, attributed to β-carotene inside the cell wall. The Raman spectra showed that the APPJ treatment of N. crassa spores in water caused degradation of β-carotene, affecting the viability of spores.

Roles of the Bacillus anthracis Spore Protein ExsK in Exosporium Maturation and Germination

DTIC Science & Technology

2009-12-01

exosporium maturation and assembly and suggest a novel role for the exosporium in germination. During starvation, bacteria of the genus Bacillus...Bacillus subtilis, the outermost struc- ture is a protective layer called the coat, which guards the spore against reactive small molecules, degradative ...analysis. Generation of anti-ExsK antibodies. Recombinant ExsK was generated and purified using the pET expression system (Novagen) according to the

Chromosome mechanics of fungi under spaceflight conditions--tetrad analysis of two-factor crosses between spore color mutants of Sordaria macrospora.

PubMed

Hahn, A; Hock, B

1999-01-01

Spore color mutants of the fungus Sordaria macrospora Auersw. were crossed under spaceflight conditions on the space shuttle to MIR mission S/MM 05 (STS-81). The arrangement of spores of different colors in the asci allowed conclusions on the influence of spaceflight conditions on sexual recombination in fungi. Experiments on a 1-g centrifuge in space and in parallel on the ground were used for controls. The samples were analyzed microscopically on their return to earth. Each fruiting body was assessed separately. Statistical analysis of the data showed a significant increase in gene recombination frequencies caused by the heavy ion particle stream in space radiation. The lack of gravity did not influence crossing-over frequencies. Hyphae of the flown samples were assessed for DNA strand breaks. No increase in damage was found compared with the ground samples. It was shown that S. macrospora is able to repair radiation-induced DNA strand breaks within hours.

A Statistical Treatment of Bioassay Pour Fractions

NASA Technical Reports Server (NTRS)

Barengoltz, Jack; Hughes, David W.

2014-01-01

The binomial probability distribution is used to treat the statistics of a microbiological sample that is split into two parts, with only one part evaluated for spore count. One wishes to estimate the total number of spores in the sample based on the counts obtained from the part that is evaluated (pour fraction). Formally, the binomial distribution is recharacterized as a function of the observed counts (successes), with the total number (trials) an unknown. The pour fraction is the probability of success per spore (trial). This distribution must be renormalized in terms of the total number. Finally, the new renormalized distribution is integrated and mathematically inverted to yield the maximum estimate of the total number as a function of a desired level of confidence ( P(

Multi-functional nano silver: A novel disruptive and theranostic agent for pathogenic organisms in real-time

PubMed Central

Gopinath, Ponnusamy Manogaran; Ranjani, Anandan; Dhanasekaran, Dharumadurai; Thajuddin, Nooruddin; Archunan, Govindaraju; Akbarsha, Mohammad Abdulkader; Gulyás, Balázs; Padmanabhan, Parasuraman

2016-01-01

The present study was aimed at evaluating the fluorescence property, sporicidal potency against Bacillus and Clostridium endospores, and surface disinfecting ability of biogenic nano silver. The nano silver was synthesized using an actinobacterial cell-filtrate. The fluorescence property as well as imaging facilitator potency of this nano silver was verified adopting spectrofluorometer along with fluorescent and confocal laser scanning microscope wherein strong emission and bright green fluorescence, respectively, on the entire spore surface was observed. Subsequently, the endospores of B. subtilis, B. cereus, B. amyloliquefaciens, C. perfringens and C. difficile were treated with physical sporicides, chemical sporicides and nano silver, in which the nano silver brought about pronounced inhibition even at a very low concentration. Finally, the environmental surface-sanitizing potency of nano silver was investigated adopting cage co-contamination assay, wherein vital organs of mice exposed to the nano silver-treated cage did not show any signs of pathological lesions, thus signifying the ability of nano silver to completely disinfect the spore or reduce the count required for infection. Taken these observations together, we have shown the multi-functional biological properties of the nano silver, synthesized using an actinobacterial cell-filtrate, which could be of application in advanced diagnostics, biomedical engineering and therapeutics in the near future. PMID:27666290

The differential effects of heat-shocking on the viability of spores from Bacillus anthracis, Bacillus subtilis, and Clostridium sporogenes after treatment with peracetic acid- and glutaraldehyde-based disinfectants.

PubMed

March, Jordon K; Pratt, Michael D; Lowe, Chinn-Woan; Cohen, Marissa N; Satterfield, Benjamin A; Schaalje, Bruce; O'Neill, Kim L; Robison, Richard A

2015-10-01

This study investigated (1) the susceptibility of Bacillus anthracis (Ames strain), Bacillus subtilis (ATCC 19659), and Clostridium sporogenes (ATCC 3584) spores to commercially available peracetic acid (PAA)- and glutaraldehyde (GA)-based disinfectants, (2) the effects that heat-shocking spores after treatment with these disinfectants has on spore recovery, and (3) the timing of heat-shocking after disinfectant treatment that promotes the optimal recovery of spores deposited on carriers. Suspension tests were used to obtain inactivation kinetics for the disinfectants against three spore types. The effects of heat-shocking spores after disinfectant treatment were also determined. Generalized linear mixed models were used to estimate 6-log reduction times for each spore type, disinfectant, and heat treatment combination. Reduction times were compared statistically using the delta method. Carrier tests were performed according to AOAC Official Method 966.04 and a modified version that employed immediate heat-shocking after disinfectant treatment. Carrier test results were analyzed using Fisher's exact test. PAA-based disinfectants had significantly shorter 6-log reduction times than the GA-based disinfectant. Heat-shocking B. anthracis spores after PAA treatment resulted in significantly shorter 6-log reduction times. Conversely, heat-shocking B. subtilis spores after PAA treatment resulted in significantly longer 6-log reduction times. Significant interactions were also observed between spore type, disinfectant, and heat treatment combinations. Immediately heat-shocking spore carriers after disinfectant treatment produced greater spore recovery. Sporicidal activities of disinfectants were not consistent across spore species. The effects of heat-shocking spores after disinfectant treatment were dependent on both disinfectant and spore species. Caution must be used when extrapolating sporicidal data of disinfectants from one spore species to another. Heat-shocking provides a more accurate picture of spore survival for only some disinfectant/spore combinations. Collaborative studies should be conducted to further examine a revision of AOAC Official Method 966.04 relative to heat-shocking. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

New amino acid germinants for spores of the enterotoxigenic Clostridium perfringens type A isolates.

PubMed

Udompijitkul, Pathima; Alnoman, Maryam; Banawas, Saeed; Paredes-Sabja, Daniel; Sarker, Mahfuzur R

2014-12-01

Clostridium perfringens spore germination plays a critical role in the pathogenesis of C. perfringens-associated food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases. Germination is initiated when bacterial spores sense specific nutrient germinants (such as amino acids) through germinant receptors (GRs). In this study, we aimed to identify and characterize amino acid germinants for spores of enterotoxigenic C. perfringens type A. The polar, uncharged amino acids at pH 6.0 efficiently induced germination of C. perfringens spores; L-asparagine, L-cysteine, L-serine, and L-threonine triggered germination of spores of most FP and NFB isolates; whereas, L-glutamine was a unique germinant for FP spores. For cysteine- or glutamine-induced germination, gerKC spores (spores of a gerKC mutant derivative of FP strain SM101) germinated to a significantly lower extent and released less DPA than wild type spores; however, a less defective germination phenotype was observed in gerAA or gerKB spores. The germination defects in gerKC spores were partially restored by complementing the gerKC mutant with a recombinant plasmid carrying wild-type gerKA-KC, indicating that GerKC is an essential GR protein. The gerKA, gerKC, and gerKB spores germinated significantly slower with L-serine and L-threonine than their parental strain, suggesting the requirement for these GR proteins for normal germination of C. perfringens spores. In summary, these results indicate that the polar, uncharged amino acids at pH 6.0 are effective germinants for spores of C. perfringens type A and that GerKC is the main GR protein for germination of spores of FP strain SM101 with L-cysteine, L-glutamine, and L-asparagine. Copyright © 2014 Elsevier Ltd. All rights reserved.

Influence of spore moisture content on the dry-heat resistance of Bacillus subtilis var. niger.

PubMed

Angelotti, R; Maryanski, J H; Butler, T F; Peeler, J T; Campbell, J E

1968-05-01

The dry-heat resistance of Bacillus subtilis var. niger spores located in or on various materials was determined as D and z values in the range of 105 through 160 C. The systems tested included spores located on steel and paper strips, spores located between stainless-steel washers mated together under 150 inch-lb and 12 inch-lb of torque, and spores encapsulated in methylmethacrylate and epoxy plastics. D values for a given temperature varied with the test system. High D values were observed for the systems in which spores were encapsulated or under heavy torque, whereas lower D values were observed for the steel and paper strip systems and the lightly torqued system. Similar z values were obtained for the plastic and steel strip systems (z(D) = 21 C), but an unusually low z for spores on paper (z(D) = 12.9 C) and an unusually high z for spores on steel washers mated at 150 inch-lb of torque (z(D) = 32 C) were observed. The effect of spore moisture content on the D value of spores encapsulated in water-impermeable plastic was determined, and maximal resistance was observed for spores with a water activity (a(w)) of 0.2 to 0.4. Significantly decreased D values were observed for spores with moisture contents below a(w) 0.2 or above a(w) 0.4. The data indicate that the important factors to be considered when measuring the dry heat resistance of spores are (i) the initial moisture content of the spore, (ii) the rate of spore desiccation during heating, (iii) the water retention capacity of the material in or on which spores are located, and (iv) the relative humidity of the system at the test temperature.

Sensitizing Clostridium difficile Spores with Germinants on Skin and Environmental Surfaces Represents a New Strategy for Reducing Spores via Ambient Mechanisms

PubMed Central

Nerandzic, Michelle M.; Donskey, Curtis J.

2017-01-01

Background Clostridium difficile is a leading cause of healthcare-associated infections worldwide. Prevention of C. difficile transmission is challenging because spores are not killed by alcohol-based hand sanitizers or many commonly used disinfectants. One strategy to control spores is to induce germination, thereby rendering the spores more susceptible to benign disinfection measures and ambient stressors. Methods/Results C. difficile spores germinated on skin after a single application of cholic acid-class bile salts and co-germinants; for 4 C. difficile strains, recovery of viable spores from skin was reduced by ~0.3 log10CFU to 2 log10CFU after 2 hours and ~1 log10CFU to > 2.5 log10CFU after 24 hours. The addition of taurocholic acid to 70% and 30% ethanol significantly enhanced reduction of viable spores on skin and on surfaces. Desiccation, and to a lesser extent the presence of oxygen, were identified as the stressors responsible for reductions of germinated spores on skin and surfaces. Additionally, germinated spores became susceptible to killing by pH 1.5 hydrochloric acid, suggesting that germinated spores that remain viable on skin and surfaces might be killed by gastric acid after ingestion. Antibiotic-treated mice did not become colonized after exposure to germinated spores, whereas 100% of mice became colonized after exposure to the same quantity of dormant spores. Conclusions Germination could provide a new approach to reduce C. difficile spores on skin and in the environment and to render surviving spores less capable of causing infection. Our findings suggest that it may be feasible to develop alcohol-based hand sanitizers containing germinants that reduce spores on hands. PMID:29167835

Imaging bacterial spores by soft-x-ray microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stead, A.D.; Ford, T.W.; Judge, J.

1997-04-01

Bacterial spores are able to survive dehydration, but neither the physiological nor structural basis of this have been fully elucidated. Furthermore, once hydrated, spores often require activation before they will germinate. Several treatments can be used to activate spores, but in the case of Bacillus subtlis the most effective is heat treatment. The physiological mechanism associated with activation is also not understood, but some workers suggest that the loss of calcium from the spores may be critical. However, just prior to germination, the spores change from being phase bright to phase dark when viewed by light microscopy. Imaging spores bymore » soft x-ray microscopy is possible without fixation. Thus, in contrast to electron microscopy, it is possible to compare the structure of dehydrated and hydrated spores in a manner not possible previously. A further advantage is that it is possible to monitor individual spores by phase contrast light microscopy immediately prior to imaging with soft x-rays; whereas, with both electron microscopy and biochemical studies, it is a population of spores being studied without knowledge of the phase characteristics of individual spores. This study has therefore tried to compare dehydrated and hydrated spores and to determine if there is a mass loss from individual spores as they pass the transition from being phase bright to phase dark.« less

Morphogenetic Pathway of Spore Wall Assembly in Saccharomyces cerevisiae

PubMed Central

Coluccio, Alison; Bogengruber, Edith; Conrad, Michael N.; Dresser, Michael E.; Briza, Peter; Neiman, Aaron M.

2004-01-01

The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation. A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor. The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays. Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly. The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins. The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure. PMID:15590821

Inactivation of Bacillus subtilis spores by high pressure CO2 with high temperature.

PubMed

Rao, Lei; Xu, Zhenzhen; Wang, Yongtao; Zhao, Feng; Hu, Xiaosong; Liao, Xiaojun

2015-07-16

The objective of this study was to investigate the inactivation of the Bacillus subtilis spores by high pressure CO2 combined with high temperature (HPCD+HT) and to analyze the clumping effect of the spores on their HPCD+HT resistance. The spores of B. subtilis were subjected to heat at 0.1 MPa and HPCD at 6.5-25 MPa, and 82 °C, 86 °C, and 91 °C for 0-120 min. The spores were effectively inactivated by HPCD+HT, but a protective effect on the spores was also found, which was closely correlated to the pressure, temperature and time. The spores treated by HPCD+HT at 6.5 and 10 MPa exhibited a two-stage inactivation curve of shoulder and log-linear regions whereas the spores at 15-25 MPa exhibited a three-stage inactivation curve of shoulder, log-linear and tailing regions, and these curves were well fitted to the Geeraerd model. Approximately 90% of pyridine-2,6-dicarboxylic acid (DPA) was released after HPCD+HT and the 90% DPA release time depend on the pressure and temperature. Moreover, the spore clumping in suspensions was examined by dynamic light scattering. The particle size of the spore suspensions increased with the increase of pressure, temperature and time, indicating the spore clumping. 0.1% Tween 80 as a surfactant inhibited the spore clumping and increased the inactivation ratio of the spores by HPCD+HT. These results indicated that the spore clumping enhanced the spores' resistance to HPCD+HT and induced a protective effect. Copyright © 2015 Elsevier B.V. All rights reserved.

Monitoring of Commitment, Blocking, and Continuation of Nutrient Germination of Individual Bacillus subtilis Spores

PubMed Central

Zhang, Pengfei; Liang, Jintao; Yi, Xuan; Setlow, Peter

2014-01-01

Short exposures of Bacillus spores to nutrient germinants can commit spores to germinate when germinants are removed or their binding to the spores' nutrient germinant receptors (GRs) is inhibited. Bacillus subtilis spores were exposed to germinants for various periods, followed by germinant removal to prevent further commitment. Release of spore dipicolinic acid (DPA) was then measured by differential interference contrast microscopy to monitor germination of multiple individual spores, and spores did not release DPA after 1 to 2 min of germinant exposure until ∼7 min after germinant removal. With longer germinant exposures, percentages of committed spores with times for completion of DPA release (Trelease) greater than the time of germinant removal (Tb) increased, while the time Tlag − Tb, where Tlag represents the time when rapid DPA release began, was decreased but rapid DPA release times (ΔTrelease = Trelease − Tlag) were increased; Factors affecting average Trelease values and the percentages of committed spores were germinant exposure time, germinant concentration, sporulation conditions, and spore heat activation, as previously shown for commitment of spore populations. Surprisingly, germination of spores given a 2nd short germinant exposure 30 to 45 min after a 1st exposure of the same duration was significantly higher than after the 1st exposure, but the number of spores that germinated in the 2nd germinant exposure decreased as the interval between germinant exposures increased up to 12 h. The latter results indicate that spores have some memory, albeit transient, of their previous exposure to nutrient germinants. PMID:24769693

Recombinant lipoprotein-based vaccine candidates against C. difficile infections.

PubMed

Huang, Jui-Hsin; Wu, Chia-Wei; Lien, Shu-Pei; Leng, Chih-Hsiang; Hsiao, Kuang-Nan; Liu, Shih-Jen; Chen, Hsin-Wei; Siu, Leung-Kei; Chong, Pele

2015-08-07

Opportunistically nosocomial infections in hospitalized patients are often related to Clostridium difficile infections (CDI) due to disruption of the intestinal micro-flora by antibiotic therapies during hospitalization. Clostridial exotoxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) in the host intestine, disrupt the intestinal barrier leading to acute inflammation and diarrhea. The C-terminal receptor binding domain of TcdA (A-rRBD) has been shown to elicit antibody responses that neutralize TcdA toxicity in Vero cell cytotoxicity assays, but not effectively protect hamsters against a lethal dose challenge of C. difficile spores. To develop an effective recombinant subunit vaccine against CDI, A-rRBD was lipidated (rlipoA-RBD) as a rational design to contain an intrinsic adjuvant, a toll-like receptor 2 agonist and expressed in Escherichia coli. The purified rlipoA-RBD was characterized immunologically and found to have the following properties: (a) mice, hamsters and rabbits vaccinated with 3 μg of rlipoA-RBD produced strong antibody responses that neutralized TcdA toxicity in Vero cell cytotoxicity assays; furthermore, the neutralization titer was comparable to those obtained from antisera immunized either with 10 μg of TcdA toxoid or 30 μg of A-rRBD; (b) rlipoA-RBD elicited immune responses and protected mice from TcdA challenge, but offered insignificant protection (10 to 20 %) against C. difficile spores challenge in hamster models; (c) only rlipoA-RBD formulated with B-rRBD consistently confers protection (90 to 100 %) in the hamster challenge model; and (d) rlipoA-RBD was found to be 10-fold more potent than A-rRBD as an adjuvant to enhancing immune responses against a poor antigen such as ovalbumin. These results indicate that rlipoA-RBD formulated with B-rRBD could be an excellent vaccine candidate for preclinical studies and future clinical trials.

A Modified R-Type Bacteriocin Specifically Targeting Clostridium difficile Prevents Colonization of Mice without Affecting Gut Microbiota Diversity

PubMed Central

Gebhart, Dana; Lok, Stephen; Clare, Simon; Tomas, Myreen; Stares, Mark; Scholl, Dean; Donskey, Curtis J.; Lawley, Trevor D.

2015-01-01

ABSTRACT Clostridium difficile is a leading cause of nosocomial infections worldwide and has become an urgent public health threat requiring immediate attention. Epidemic lineages of the BI/NAP1/027 strain type have emerged and spread through health care systems across the globe over the past decade. Limiting person-to-person transmission and eradicating C. difficile, especially the BI/NAP1/027 strain type, from health care facilities are difficult due to the abundant shedding of spores that are impervious to most interventions. Effective prophylaxis for C. difficile infection (CDI) is lacking. We have genetically modified a contractile R-type bacteriocin (“diffocin”) from C. difficile strain CD4 to kill BI/NAP1/027-type strains for this purpose. The natural receptor binding protein (RBP) responsible for diffocin targeting was replaced with a newly discovered RBP identified within a prophage of a BI/NAP1/027-type target strain by genome mining. The resulting modified diffocins (a.k.a. Avidocin-CDs), Av-CD291.1 and Av-CD291.2, were stable and killed all 16 tested BI/NAP1/027-type strains. Av-CD291.2 administered in drinking water survived passage through the mouse gastrointestinal (GI) tract, did not detectably alter the mouse gut microbiota or disrupt natural colonization resistance to C. difficile or the vancomycin-resistant Enterococcus faecium (VREF), and prevented antibiotic-induced colonization of mice inoculated with BI/NAP1/027-type spores. Given the high incidence and virulence of the pathogen, preventing colonization by BI/NAP1/027-type strains and limiting their transmission could significantly reduce the occurrence of the most severe CDIs. This modified diffocin represents a prototype of an Avidocin-CD platform capable of producing targetable, precision anti-C. difficile agents that can prevent and potentially treat CDIs without disrupting protective indigenous microbiota. PMID:25805733

Bacillus spore-based oral carriers loading curcumin for the therapy of colon cancer.

PubMed

Yin, Liang; Meng, Zhan; Zhang, Yuxiao; Hu, Kaikai; Chen, Wuya; Han, Kaibin; Wu, Bao-Yan; You, Rong; Li, Chu-Hua; Jin, Ying; Guan, Yan-Qing

2018-02-10

Oral drug delivery has attracted substantial attention due to its advantages over other administration routes. Bacillus spores, as oral probiotic agents, are applied widely. In this paper, a novel Bacillus spore-based oral colon targeted carrier loading curcumin was developed for colon cancer treatment. Curcumin was linked covalently with the outer coat of Bacillus spore and folate, respectively (SPORE-CUR-FA). Bacillus spores are capable of delivering drugs to the colon area through gastric barrier, taking the advantage of its tolerance to the harsh conditions and disintegration of the outer coat of spores after germination in the colon. The drug release in vitro and in vivo of SPORE-CUR-FA was investigated. Results showed that SPORE-CUR-FA had the characteristics of colon-targeted drug release. Pharmacokinetic studies confirmed that Bacillus spore-based carriers could efficiently improve the oral bioavailability of curcumin. In vitro and in vivo anti-tumor studies showed that SPORE-CUR-FA had substantial ability for inhibiting colon cancer cells. These findings suggest that this Bacillus spore-based oral drug delivery system has a great potential for the treatment of colon cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Spore formation in Myxococcus xanthus is tied to cytoskeleton functions and polysaccharide spore coat deposition

PubMed Central

Müller, Frank D.; Schink, Christian W.; Hoiczyk, Egbert; Cserti, Emöke; Higgs, Penelope I.

2011-01-01

Summary Myxococcus xanthus is a Gram-negative bacterium that differentiates into environmentally resistant spores. Spore differentiation involves septation-independent remodelling of the rod-shaped vegetative cell into a spherical spore and deposition of a thick and compact spore coat outside of the outer membrane. Our analyses suggest that spore coat polysaccharides are exported to the cell surface by the Exo outer membrane polysaccharide export/polysaccharide co-polymerase 2a (OPX/PCP-2a) machinery. Conversion of the capsule-like polysaccharide layer into a compact spore coat layer requires the Nfs proteins which likely form a complex in the cell envelope. Mutants in either nfs, exo, or two other genetic loci encoding homologs of polysaccharide synthesis enzymes, fail to complete morphogenesis from rods to spherical spores and instead produce a transient state of deformed cell morphology before reversion into typical rods. We additionally provide evidence that the cell cytoskeletal protein, MreB, plays an important role in rod to spore morphogenesis and for spore outgrowth. These studies provide evidence that this novel gram-negative differentiation process is tied to cytoskeleton functions and polysaccharide spore coat deposition. PMID:22188356

Spore ornamentation of Haplosporidium pickfordi Barrow, 1961 (Haplosporidia), a parasite of freshwater snails in Michigan, USA.

PubMed

Burreson, E M

2001-01-01

Spore ornamentation is increasingly recognized as a key character for species differentiation and genus assignment in the phylum Haplosporidia. Unfortunately, spore ornamentation is known for only a small number of described species so it is difficult to assign most species to genera with any confidence. Scanning and transmission electron microscopy were used to determine the presence and morphology of spore ornamentation of Haplosporidium pickfordi collected from the digestive gland of the snail Physella parkeri in Douglas Lake, Michigan. Spores possess filaments that are derived from the spore wall and originate from two separate areas at the posterior end of the spore. When spores are first isolated from host tissue, filaments are fused into a sheet that wraps around the spore, passing under the opercular lid. These filaments gradually unravel when spores are held in water and after about 14 d most filaments project freely from the posterior end of the spore. The number of filaments could not be determined with certainty, but appears to be approximately nine. Filaments are 100 nm in diam. and up to 50 microm in length. The presence of spore wall-derived filaments confirms the placement of the parasite in the genus Haplosporidium.

Fruiting Body Formation in Volvariella volvacea Can Occur Independently of Its MAT-A-Controlled Bipolar Mating System, Enabling Homothallic and Heterothallic Life Cycles.

PubMed

Chen, Bingzhi; van Peer, Arend F; Yan, Junjie; Li, Xiao; Xie, Bin; Miao, Juan; Huang, Qianhui; Zhang, Lei; Wang, Wei; Fu, Junsheng; Zhang, Xiang; Zhang, Xiaoyin; Hu, Fengli; Kong, Qingfang; Sun, Xianyun; Zou, Feng; Zhang, Hanxing; Li, Shaojie; Xie, Baogui

2016-07-07

Volvariella volvacea is an important crop in Southeast Asia, but erratic fruiting presents a serious challenge for its production and breeding. Efforts to explain inconsistent fruiting have been complicated by the multinucleate nature, typical lack of clamp connections, and an incompletely identified sexual reproductive system. In this study, we addressed the life cycle of V. volvacea using whole genome sequencing, cloning of MAT loci, karyotyping of spores, and fruiting assays. Microscopy analysis of spores had previously indicated the possible coexistence of heterothallic and homothallic life cycles. Our analysis of the MAT loci showed that only MAT-A, and not MAT-B, controlled heterokaryotization. Thus, the heterothallic life cycle was bipolar. Karyotyping of single spore isolates (SSIs) using molecular markers supported the existence of heterokaryotic spores. However, most SSIs were clearly not heterokaryotic, yet contained structural variation (SV) markers relating to both alleles of both parents. Heterokaryons from crossed, self-sterile homokaryons could produce fruiting bodies, agreeing with bipolar heterothallism. Meanwhile, some SSIs with two different MAT-A loci also produced fruiting bodies, which supported secondary homothallism. Next, SSIs that clearly contained only one MAT-A locus (homothallism) were also able to fruit, demonstrating that self-fertile SSIs were not, per definition, secondary homothallic, and that a third life cycle or genetic mechanism must exist. Finally, recombination between SV markers was normal, yet 10 out of 24 SV markers showed 1:2 or 1:3 distributions in the spores, and large numbers of SSIs contained doubled SV markers. This indicated selfish genes, and possibly partial aneuploidy. Copyright © 2016 Chen et al.

High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis.

PubMed

Meeske, Alexander J; Rodrigues, Christopher D A; Brady, Jacqueline; Lim, Hoong Chuin; Bernhardt, Thomas G; Rudner, David Z

2016-01-01

The differentiation of the bacterium Bacillus subtilis into a dormant spore is among the most well-characterized developmental pathways in biology. Classical genetic screens performed over the past half century identified scores of factors involved in every step of this morphological process. More recently, transcriptional profiling uncovered additional sporulation-induced genes required for successful spore development. Here, we used transposon-sequencing (Tn-seq) to assess whether there were any sporulation genes left to be discovered. Our screen identified 133 out of the 148 genes with known sporulation defects. Surprisingly, we discovered 24 additional genes that had not been previously implicated in spore formation. To investigate their functions, we used fluorescence microscopy to survey early, middle, and late stages of differentiation of null mutants from the B. subtilis ordered knockout collection. This analysis identified mutants that are delayed in the initiation of sporulation, defective in membrane remodeling, and impaired in spore maturation. Several mutants had novel sporulation phenotypes. We performed in-depth characterization of two new factors that participate in cell-cell signaling pathways during sporulation. One (SpoIIT) functions in the activation of σE in the mother cell; the other (SpoIIIL) is required for σG activity in the forespore. Our analysis also revealed that as many as 36 sporulation-induced genes with no previously reported mutant phenotypes are required for timely spore maturation. Finally, we discovered a large set of transposon insertions that trigger premature initiation of sporulation. Our results highlight the power of Tn-seq for the discovery of new genes and novel pathways in sporulation and, combined with the recently completed null mutant collection, open the door for similar screens in other, less well-characterized processes.

High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis

PubMed Central

Brady, Jacqueline; Lim, Hoong Chuin; Bernhardt, Thomas G.; Rudner, David Z.

2016-01-01

The differentiation of the bacterium Bacillus subtilis into a dormant spore is among the most well-characterized developmental pathways in biology. Classical genetic screens performed over the past half century identified scores of factors involved in every step of this morphological process. More recently, transcriptional profiling uncovered additional sporulation-induced genes required for successful spore development. Here, we used transposon-sequencing (Tn-seq) to assess whether there were any sporulation genes left to be discovered. Our screen identified 133 out of the 148 genes with known sporulation defects. Surprisingly, we discovered 24 additional genes that had not been previously implicated in spore formation. To investigate their functions, we used fluorescence microscopy to survey early, middle, and late stages of differentiation of null mutants from the B. subtilis ordered knockout collection. This analysis identified mutants that are delayed in the initiation of sporulation, defective in membrane remodeling, and impaired in spore maturation. Several mutants had novel sporulation phenotypes. We performed in-depth characterization of two new factors that participate in cell–cell signaling pathways during sporulation. One (SpoIIT) functions in the activation of σE in the mother cell; the other (SpoIIIL) is required for σG activity in the forespore. Our analysis also revealed that as many as 36 sporulation-induced genes with no previously reported mutant phenotypes are required for timely spore maturation. Finally, we discovered a large set of transposon insertions that trigger premature initiation of sporulation. Our results highlight the power of Tn-seq for the discovery of new genes and novel pathways in sporulation and, combined with the recently completed null mutant collection, open the door for similar screens in other, less well-characterized processes. PMID:26735940

Roles of germination-specific lytic enzymes CwlJ and SleB in Bacillus anthracis.

PubMed

Heffron, Jared D; Orsburn, Benjamin; Popham, David L

2009-04-01

The structural characteristics of a spore enable it to withstand stresses that typically kill a vegetative cell. Spores remain dormant until small molecule signals induce them to germinate into vegetative bacilli. Germination requires degradation of the thick cortical peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four putative GSLEs, based upon sequence similarities with enzymes in other species: SleB, CwlJ1, CwlJ2, and SleL. In this study, the roles of SleB, CwlJ1, and CwlJ2 were examined. The expression levels of all three genes peak 3.5 h into sporulation. Genetic analysis revealed that, similar to other known GSLEs, none of these gene products are individually required for growth, sporulation, or triggering of germination. However, later germination events are affected in spores lacking CwlJ1 or SleB. Compared to the wild type, germinating spores without CwlJ1 suffer a delay in optical density loss and cortex peptidoglycan release. The absence of SleB also causes a delay in cortex fragment release. A double mutant lacking both SleB and CwlJ1 is completely blocked in cortex hydrolysis and progresses through outgrowth to produce colonies at a frequency 1,000-fold lower than that of the wild-type strain. A null mutation eliminating CwlJ2 has no effect on germination. High-performance liquid chromatography and mass spectroscopy analysis revealed that SleB is required for lytic transglycosylase activity. CwlJ1 also clearly participates in cortex hydrolysis, but its specific mode of action remains unclear. Understanding the lytic germination activities that naturally diminish spore resistance can lead to methods for prematurely inducing them, thus simplifying the process of treating contaminated sites.

Mold and Endotoxin Levels in the Aftermath of Hurricane Katrina: A Pilot Project of Homes in New Orleans Undergoing Renovation

PubMed Central

Chew, Ginger L.; Wilson, Jonathan; Rabito, Felicia A.; Grimsley, Faye; Iqbal, Shahed; Reponen, Tiina; Muilenberg, Michael L.; Thorne, Peter S.; Dearborn, Dorr G.; Morley, Rebecca L.

2006-01-01

Background After Hurricane Katrina, many New Orleans homes remained flooded for weeks, promoting heavy microbial growth. Objectives A small demonstration project was conducted November 2005–January 2006 aiming to recommend safe remediation techniques and safe levels of worker protection, and to characterize airborne mold and endotoxin throughout cleanup. Methods Three houses with floodwater lines between 0.3 and 2 m underwent intervention, including disposal of damaged furnishings and drywall, cleaning surfaces, drying remaining structure, and treatment with a biostatic agent. We measured indoor and outdoor bioaerosols before, during, and after intervention. Samples were analyzed for fungi [culture, spore analysis, polymerase chain reaction (PCR)] and endotoxin. In one house, real-time particle counts were also assessed, and respirator-efficiency testing was performed to establish workplace protection factors (WPF). Results At baseline, culturable mold ranged from 22,000 to 515,000 colony-forming units/m3, spore counts ranged from 82,000 to 630,000 spores/m3, and endotoxin ranged from 17 to 139 endotoxin units/m3. Culture, spore analysis, and PCR indicated that Penicillium, Aspergillus, and Paecilomyces predominated. After intervention, levels of mold and endotoxin were generally lower (sometimes, orders of magnitude). The average WPF against fungal spores for elastomeric respirators was higher than for the N-95 respirators. Conclusions During baseline and intervention, mold and endotoxin levels were similar to those found in agricultural environments. We strongly recommend that those entering, cleaning, and repairing flood-damaged homes wear respirators at least as protective as elastomeric respirators. Recommendations based on this demonstration will benefit those involved in the current cleanup activities and will inform efforts to respond to future disasters. PMID:17185280

Mold and endotoxin levels in the aftermath of Hurricane Katrina: a pilot project of homes in New Orleans undergoing renovation.

PubMed

Chew, Ginger L; Wilson, Jonathan; Rabito, Felicia A; Grimsley, Faye; Iqbal, Shahed; Reponen, Tiina; Muilenberg, Michael L; Thorne, Peter S; Dearborn, Dorr G; Morley, Rebecca L

2006-12-01

After Hurricane Katrina, many New Orleans homes remained flooded for weeks, promoting heavy microbial growth. A small demonstration project was conducted November 2005-January 2006 aiming to recommend safe remediation techniques and safe levels of worker protection, and to characterize airborne mold and endotoxin throughout cleanup. Three houses with floodwater lines between 0.3 and 2 m underwent intervention, including disposal of damaged furnishings and drywall, cleaning surfaces, drying remaining structure, and treatment with a biostatic agent. We measured indoor and outdoor bioaerosols before, during, and after intervention. Samples were analyzed for fungi [culture, spore analysis, polymerase chain reaction (PCR)] and endotoxin. In one house, realtime particle counts were also assessed, and respirator-efficiency testing was performed to establish workplace protection factors (WPF). At baseline, culturable mold ranged from 22,000 to 515,000 colony-forming units/m3, spore counts ranged from 82,000 to 630,000 spores/m3, and endotoxin ranged from 17 to 139 endotoxin units/m3. Culture, spore analysis, and PCR indicated that Penicillium, Aspergillus, and Paecilomyces predominated. After intervention, levels of mold and endotoxin were generally lower (sometimes, orders of magnitude). The average WPF against fungal spores for elastomeric respirators was higher than for the N95 respirators. During baseline and intervention, mold and endotoxin levels were similar to those found in agricultural environments. We strongly recommend that those entering, cleaning, and repairing flood-damaged homes wear respirators at least as protective as elastomeric respirators. Recommendations based on this demonstration will benefit those involved in the current cleanup activities and will inform efforts to respond to future disasters.

ENZYMES OF GLUCOSE AND PYRUVATE CATABOLISM IN CELLS, SPORES, AND GERMINATED SPORES OF CLOSTRIDIUM BOTULINUM1

PubMed Central

Simmons, Richard J.; Costilow, Ralph N.

1962-01-01

Simmons, R. J. (Michigan State University, East Lansing), and R. N. Costilow. Enzymes of glucose and pyruvate catabolism in cells, spores, and germinated spores of Clostridium botulinum. J. Bacteriol. 84:1274–1281. 1962.—An investigation was made of the enzymes of vegetative cells, spores, and germinated spores of Clostridium botulinum 62-A to elucidate a pathway of glucose metabolism. Manometric studies were conducted with intact cells, and various enzymes and enzyme systems were assayed in cell-free and spore-free extracts by use of spectrophotometric and colorimetric procedures. Glucose fermentation was found to be inducible; glucokinase was the controlling enzyme. All other enzymes of the Embden-Meyerhof-Parnas (EMP) pathway were found in both induced and non-induced cells, but they were in relatively low concentrations in the latter. This, plus the fact that no glucose-6-phosphate dehydrogenase was detected, led to the conclusion that glucose is catabolized primarily by the EMP system. A number of glycolytic enzymes were also found in extracts of spores and germinated spores of this organism, but the activities were extremely low as compared with activities in cell extracts. A phosphoroclastic-type reaction was readily demonstrated in both glucose-adapted and non-adapted cells, but not in spores and germinated spores. However, both acetokinase and phosphotransacetylase, as well as coenzyme A transphorase, were detected in spores and germinated-spore extracts, although at very low activity levels as compared with cell extracts. The specific activity of diaphorase in spore extracts was about one-half that of corresponding cell extracts, and the activity of reduced diphosphopyridine nucleotide (DPNH) oxidase was actually higher in the spore extracts. In addition, the DPNH oxidase in spore extracts was considerably more heat-stable than that in extracts of cells or germinated spores. PMID:13977433

Meteorological factors associated with abundance of airborne fungal spores over natural vegetation

NASA Astrophysics Data System (ADS)

Crandall, Sharifa G.; Gilbert, Gregory S.

2017-08-01

The abundance of airborne fungal spores in agricultural and urban settings increases with greater air temperature, relative humidity, or precipitation. The same meteorological factors that affect temporal patterns in spore abundance in managed environments also vary spatially across natural habitats in association with differences in vegetation structure. Here we investigated how temporal and spatial variation in aerial spore abundance is affected by abiotic (weather) and biotic (vegetation) factors as a foundation for predicting how fungi may respond to changes in weather and land-use patterns. We measured the phenology of airborne fungal spores across a mosaic of naturally occurring vegetation types at different time scales to describe (1) how spore abundance changes over time, (2) which local meteorological variables are good predictors for airborne spore density, and (3) whether spore abundance differs across vegetation types. Using an air volumetric vacuum sampler, we collected spore samples at 3-h intervals over a 120-h period in a mixed-evergreen forest and coastal prairie to measure diurnal, nocturnal, and total airborne spore abundance across vegetation types. Spore samples were also collected at weekly and monthly intervals in mixed-evergreen forest, redwood forest, and maritime chaparral vegetation types from 12 field sites across two years. We found greater airborne spore densities during the wetter winter months compared to the drier summer months. Mean total spore abundance in the mixed-evergreen forest was twice than in the coastal prairie, but there were no significant differences in total airborne spore abundance among mixed-evergreen forest, redwood forest, and maritime chaparral vegetation types. Weekly and monthly peaks in airborne spore abundance corresponded with rain events and peaks in soil moisture. Overall, temporal patterns in meteorological factors were much more important in determining airborne fungal spore abundance than the vegetation type. This suggests that overall patterns of fungal spore dynamics may be predictable across heterogeneous landscapes based on local weather patterns.

Correlation of simulation/finite element analysis to the separation of intrinsically magnetic spores and red blood cells using a microfluidic magnetic deposition system.

PubMed

Sun, Jianxin; Moore, Lee; Xue, Wei; Kim, James; Zborowski, Maciej; Chalmers, Jeffrey J

2018-05-01

Magnetic separation of cells has been, and continues to be, widely used in a variety of applications, ranging from healthcare diagnostics to detection of food contamination. Typically, these technologies require cells labeled with antibody magnetic particle conjugate and a high magnetic energy gradient created in the flow containing the labeled cells (i.e., a column packed with magnetically inducible material), or dense packing of magnetic particles next to the flow cell. Such designs, while creating high magnetic energy gradients, are not amenable to easy, highly detailed, mathematic characterization. Our laboratories have been characterizing and developing analysis and separation technology that can be used on intrinsically magnetic cells or spores which are typically orders of magnitude weaker than typically immunomagnetically labeled cells. One such separation system is magnetic deposition microscopy (MDM) which not only separates cells, but deposits them in specific locations on slides for further microscopic analysis. In this study, the MDM system has been further characterized, using finite element and computational fluid mechanics software, and separation performance predicted, using a model which combines: 1) the distribution of the intrinsic magnetophoretic mobility of the cells (spores); 2) the fluid flow within the separation device; and 3) accurate maps of the values of the magnetic field (max 2.27 T), and magnetic energy gradient (max of 4.41 T 2 /mm) within the system. Guided by this model, experimental studies indicated that greater than 95% of the intrinsically magnetic Bacillus spores can be separated with the MDM system. Further, this model allows analysis of cell trajectories which can assist in the design of higher throughput systems. © 2018 Wiley Periodicals, Inc.

The distribution of Paenibacillus larvae spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee (Apis mellifera) colonies.

PubMed

Lindström, Anders; Korpela, Seppo; Fries, Ingemar

2008-09-01

Within colony transmission of Paenibacillus larvae spores was studied by giving spore-contaminated honey comb or comb containing 100 larvae killed by American foulbrood to five experimental colonies respectively. We registered the impact of the two treatments on P. larvae spore loads in adult bees and honey and on larval mortality by culturing for spores in samples of adult bees and honey, respectively, and by measuring larval survival. The results demonstrate a direct effect of treatment on spore levels in adult bees and honey as well as on larval mortality. Colonies treated with dead larvae showed immediate high spore levels in adult bee samples, while the colonies treated with contaminated honey showed a comparable spore load but the effect was delayed until the bees started to utilize the honey at the end of the flight season. During the winter there was a build up of spores in the adult bees, which may increase the risk for infection in spring. The results confirm that contaminated honey can act as an environmental reservoir of P. larvae spores and suggest that less spores may be needed in honey, compared to in diseased brood, to produce clinically diseased colonies. The spore load in adult bee samples was significantly related to larval mortality but the spore load of honey samples was not.

Use of yeast spores for microencapsulation of enzymes.

PubMed

Shi, Libing; Li, Zijie; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

2014-08-01

Here, we report a novel method to produce microencapsulated enzymes using Saccharomyces cerevisiae spores. In sporulating cells, soluble secreted proteins are transported to the spore wall. Previous work has shown that the spore wall is capable of retaining soluble proteins because its outer layers work as a diffusion barrier. Accordingly, a red fluorescent protein (RFP) fusion of the α-galactosidase, Mel1, expressed in spores was observed in the spore wall even after spores were subjected to a high-salt wash in the presence of detergent. In vegetative cells, however, the cell wall cannot retain the RFP fusion. Although the spore wall prevents diffusion of proteins, it is likely that smaller molecules, such as sugars, pass through it. In fact, spores can contain much higher α-galactosidase activity to digest melibiose than vegetative cells. When present in the spore wall, the enzyme acquires resistance to environmental stresses including enzymatic digestion and high temperatures. The outer layers of the spore wall are required to retain enzymes but also decrease accessibility of the substrates. However, mutants with mild spore wall defects can retain and stabilize the enzyme while still permitting access to the substrate. In addition to Mel1, we also show that spores can retain the invertase. Interestingly the encapsulated invertase has significantly lower activity toward raffinose than toward sucrose.This suggests that substrate selectivity could be altered by the encapsulation.

Ecology and thermal inactivation of microbes in and on interplanetary space vehicle components. [heat sensitivity of bacterial spores

NASA Technical Reports Server (NTRS)

Campbell, J. E.; Reyes, A. L.; Wehby, A. J.; Crawford, R. G.; Wimsatt, J. C.; Peeler, J. T.

1973-01-01

The mechanism for thermal inactivation of bacterial spores under moist or dry heat was studied. Experimental conditions were established relating to spore loss of heat resistance and loss of optical density as a measure of the rate and extent of germination in spore suspensions. Events occurring during germination were correlated with phase darkening (refractility and non-refractility of spores), stainability characteristics of heat and non-heat treated spores, morphological characteristics, and studies on swelling of spores by an increase in packed cell volume.

Spore development and nuclear inheritance in arbuscular mycorrhizal fungi

PubMed Central

2011-01-01

Background A conventional tenet of classical genetics is that progeny inherit half their genome from each parent in sexual reproduction instead of the complete genome transferred to each daughter during asexual reproduction. The transmission of hereditary characteristics from parents to their offspring is therefore predictable, although several exceptions are known. Heredity in microorganisms, however, can be very complex, and even unknown as is the case for coenocytic organisms such as Arbuscular Mycorrhizal Fungi (AMF). This group of fungi are plant-root symbionts, ubiquitous in most ecosystems, which reproduce asexually via multinucleate spores for which sexuality has not yet been observed. Results We examined the number of nuclei per spore of four AMF taxa using high Z-resolution live confocal microscopy and found that the number of nuclei was correlated with spore diameter. We show that AMF have the ability, through the establishment of new symbioses, to pass hundreds of nuclei to subsequent generations of multinucleated spores. More importantly, we observed surprising heterogeneity in the number of nuclei among sister spores and show that massive nuclear migration and mitosis are the mechanisms by which AMF spores are formed. We followed spore development of Glomus irregulare from hyphal swelling to spore maturity and found that the spores reached mature size within 30 to 60 days, and that the number of nuclei per spores increased over time. Conclusions We conclude that the spores used for dispersal of AMF contain nuclei with two origins, those that migrate into the spore and those that arise by mitosis in the spore. Therefore, these spores do not represent a stage in the life cycle with a single nucleus, raising the possibility that AMF, unlike all other known eukaryotic organisms, lack the genetic bottleneck of a single-nucleus stage. PMID:21349193

Spore development and nuclear inheritance in arbuscular mycorrhizal fungi.

PubMed

Marleau, Julie; Dalpé, Yolande; St-Arnaud, Marc; Hijri, Mohamed

2011-02-24

A conventional tenet of classical genetics is that progeny inherit half their genome from each parent in sexual reproduction instead of the complete genome transferred to each daughter during asexual reproduction. The transmission of hereditary characteristics from parents to their offspring is therefore predictable, although several exceptions are known. Heredity in microorganisms, however, can be very complex, and even unknown as is the case for coenocytic organisms such as Arbuscular Mycorrhizal Fungi (AMF). This group of fungi are plant-root symbionts, ubiquitous in most ecosystems, which reproduce asexually via multinucleate spores for which sexuality has not yet been observed. We examined the number of nuclei per spore of four AMF taxa using high Z-resolution live confocal microscopy and found that the number of nuclei was correlated with spore diameter. We show that AMF have the ability, through the establishment of new symbioses, to pass hundreds of nuclei to subsequent generations of multinucleated spores. More importantly, we observed surprising heterogeneity in the number of nuclei among sister spores and show that massive nuclear migration and mitosis are the mechanisms by which AMF spores are formed. We followed spore development of Glomus irregulare from hyphal swelling to spore maturity and found that the spores reached mature size within 30 to 60 days, and that the number of nuclei per spores increased over time. We conclude that the spores used for dispersal of AMF contain nuclei with two origins, those that migrate into the spore and those that arise by mitosis in the spore. Therefore, these spores do not represent a stage in the life cycle with a single nucleus, raising the possibility that AMF, unlike all other known eukaryotic organisms, lack the genetic bottleneck of a single-nucleus stage.

Measuring spore settling velocity for an improved assessment of dispersal rates in mosses

PubMed Central

Zanatta, Florian; Patiño, Jairo; Lebeau, Frederic; Massinon, Mathieu; Hylander, Kristofer; de Haan, Myriam; Ballings, Petra; Degreef, Jerôme; Vanderpoorten, Alain

2016-01-01

Background and Aims The settling velocity of diaspores is a key parameter for the measurement of dispersal ability in wind-dispersed plants and one of the most relevant parameters in explicit dispersal models, but remains largely undocumented in bryophytes. The settling velocities of moss spores were measured and it was determined whether settling velocities can be derived from spore diameter using Stokes’ Law or if specific traits of spore ornamentation cause departures from theoretical expectations. Methods A fall tower design combined with a high-speed camera was used to document spore settling velocities in nine moss species selected to cover the range of spore diameters within the group. Linear mixed effect models were employed to determine whether settling velocity can be predicted from spore diameter, taking specific variation in shape and surface roughness into account. Key Results Average settling velocity of moss spores ranged from 0·49 to 8·52 cm s–1. There was a significant positive relationship between spore settling velocity and size, but the inclusion of variables of shape and texture of spores in the best-fit models provides evidence for their role in shaping spore settling velocities. Conclusions Settling velocities in mosses can significantly depart from expectations derived from Stokes’ Law. We suggest that variation in spore shape and ornamentation affects the balance between density and drag, and results in different dispersal capacities, which may be correlated with different life-history traits or ecological requirements. Further studies on spore ultrastructure would be necessary to determine the role of complex spore ornamentation patterns in the drag-to-mass ratio and ultimately identify what is the still poorly understood function of the striking and highly variable ornamentation patterns of the perine layer on moss spores. PMID:27296133

Microbiological efficacy of superheated steam. I. Communication: results with spores of Bacillus subtilis and Bacillus stearothermophilus and with spore earth.

PubMed

Spicher, G; Peters, J; Borchers, U

1999-02-01

For the spores of Bacillus subtilis and Bacillus stearothermophilus as well as for spore earth (acc. DIN 58,946 Part 4 of August 1982), the dependence of resistance on the superheating of the steam used to kill germs was determined. A material (glass fibre fleece) was used as the germ carrier which does not superheat on contact with steam. The temperature of the saturated steam was 100 degrees C (B. subtilis) and 120 degrees C (B. stearothermophilus and spore earth). The yardstick for the resistance of the spores or bioindicators was the exposure period of the saturated or superheated steam at which 50% of the treated test objects no longer showed any viable test germs. The spores of Bacillus subtilis were far more sensitive to superheating of steam and reacted far more than the spores of Bacillus stearothermophilus and the germs in the spore earth. When superheating by 4 Kelvin the spores of Bacillus subtilis were approximately 2.5 times more resistant than they were to saturated steam. The resistance of Bacillus stearothermophilus and spore earth was only slightly higher up to superheating by 10 Kelvin. The spores of Bacillus subtilis had the highest resistance during superheating by 29 Kelvin; they were 119 times more resistant than they were to saturated steam. The resistance maximum of the spores of Bacillus stearothermophilus was at an superheating by around 22 Kelvin. However, the spores were only 4.1 times more resistant than they were to saturated steam. When using steam to kill germs, we must expect superheated steam. This raises the question whether the spores of Bacillus stearothermophilus, with their weaker reaction to the superheating of steam, are suitable as test germs for sterilisation with steam in all cases.

False-Negative Rate and Recovery Efficiency Performance of a Validated Sponge Wipe Sampling Method

PubMed Central

Piepel, Greg F.; Boucher, Raymond; Tezak, Matt; Amidan, Brett G.; Einfeld, Wayne

2012-01-01

Recovery of spores from environmental surfaces varies due to sampling and analysis methods, spore size and characteristics, surface materials, and environmental conditions. Tests were performed to evaluate a new, validated sponge wipe method using Bacillus atrophaeus spores. Testing evaluated the effects of spore concentration and surface material on recovery efficiency (RE), false-negative rate (FNR), limit of detection (LOD), and their uncertainties. Ceramic tile and stainless steel had the highest mean RE values (48.9 and 48.1%, respectively). Faux leather, vinyl tile, and painted wood had mean RE values of 30.3, 25.6, and 25.5, respectively, while plastic had the lowest mean RE (9.8%). Results show roughly linear dependences of RE and FNR on surface roughness, with smoother surfaces resulting in higher mean REs and lower FNRs. REs were not influenced by the low spore concentrations tested (3.10 × 10−3 to 1.86 CFU/cm2). Stainless steel had the lowest mean FNR (0.123), and plastic had the highest mean FNR (0.479). The LOD90 (≥1 CFU detected 90% of the time) varied with surface material, from 0.015 CFU/cm2 on stainless steel up to 0.039 on plastic. It may be possible to improve sampling results by considering surface roughness in selecting sampling locations and interpreting spore recovery data. Further, FNR values (calculated as a function of concentration and surface material) can be used presampling to calculate the numbers of samples for statistical sampling plans with desired performance and postsampling to calculate the confidence in characterization and clearance decisions. PMID:22138998

Extracellular proteases from Streptomyces phaeopurpureus ExPro138 inhibit spore adhesion, germination and appressorium formation in Colletotrichum coccodes.

PubMed

Palaniyandi, S A; Yang, S H; Suh, J-W

2013-07-01

To study the antifungal mechanism of proteases from Streptomyces phaeopurpureus strain ExPro138 towards Colletotrichum coccodes and to evaluate its utilization as biofungicide. We screened proteolytic Streptomyces strains from the yam rhizosphere with antifungal activity. Forty proteolytic Streptomyces were isolated, among which eleven isolates showed gelatinolytic activity and antagonistic activity on C. coccodes. Of the 11 isolates, protease preparation from an isolate designated ExPro138 showed antifungal activity. 16S rDNA sequence analysis of the strain showed 99% similarity with Streptomyces phaeopurepureus (EU841588.1). Zymography analysis of the ExPro138 culture filtrate revealed that the strain produced several extracellular proteases. The protease preparation inhibited spore germination, spore adhesion to polystyrene surface and appressorium formation. Microscopic study of the interaction between ExPro138 and C. coccodes revealed that ExPro138 was mycoparasitic on C. coccodes. The protease preparation also reduced anthracnose incidence on tomato fruits compared with untreated control. This study demonstrates possibility of utilizing antifungal proteases derived from antagonistic microbes as biofungicide. Microbial proteases having the ability to inhibit spore adhesion and appressorium formation could be used to suppress infection establishment by foliar fungal pathogens at the initial stages of the infection process. Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.

Growth from spores of Clostridium perfringens in the presence of sodium nitrite.

PubMed

Labbe, R G; Duncan, C L

1970-02-01

The method by which sodium nitrite may act to prevent germination or outgrowth, or both, of heat-injured spores in canned cured meats was investigated by using Clostridium perfringens spores. Four possible mechanisms were tested: (i) prevention of germination of the heat-injured spores, (ii) prior combination with a component in a complex medium to prevent germination of heat-injured spores, (iii) inhibition of outgrowth of heat-injured spores, and (iv) induction of germination (which would render the spore susceptible to thermal inactivation). Only the third mechanism was effective with the entire spore population when levels of sodium nitrite commercially acceptable in canned cured meats were used. Concentrations of 0.02 and 0.01% prevented outgrowth of heat-sensitive and heat-resistant spores, respectively. Nitrite-induced germination occurred with higher sodium nitrite concentrations.

Spore populations among bulk tank raw milk and dairy powders are significantly different.

PubMed

Miller, Rachel A; Kent, David J; Watterson, Matthew J; Boor, Kathryn J; Martin, Nicole H; Wiedmann, Martin

2015-12-01

To accommodate stringent spore limits mandated for the export of dairy powders, a more thorough understanding of the spore species present will be necessary to develop prospective strategies to identify and reduce sources (i.e., raw materials or in-plant) of contamination. We characterized 1,523 spore isolates obtained from bulk tank raw milk (n=33 farms) and samples collected from 4 different dairy powder-processing plants producing acid whey, nonfat dry milk, sweet whey, or whey protein concentrate 80. The spores isolated comprised 12 genera, at least 44 species, and 216 rpoB allelic types. Bacillus and Geobacillus represented the most commonly isolated spore genera (approximately 68.9 and 12.1%, respectively, of all spore isolates). Whereas Bacillus licheniformis was isolated from samples collected from all plants and farms, Geobacillus spp. were isolated from samples from 3 out of 4 plants and just 1 out of 33 farms. We found significant differences between the spore population isolated from bulk tank raw milk and those isolated from dairy powder plant samples, except samples from the plant producing acid whey. A comparison of spore species isolated from raw materials and finished powders showed that although certain species, such as B. licheniformis, were found in both raw and finished product samples, other species, such as Geobacillus spp. and Anoxybacillus spp., were more frequently isolated from finished powders. Importantly, we found that 8 out of 12 genera were isolated from at least 2 different spore count methods, suggesting that some spore count methods may provide redundant information if used in parallel. Together, our results suggest that (1) Bacillus and Geobacillus are the predominant spore contaminants in a variety of dairy powders, implying that future research efforts targeted at elucidating approaches to reduce levels of spores in dairy powders should focus on controlling levels of spore isolates from these genera; and (2) the spore populations isolated from bulk tank raw milk and some dairy powder products are significantly different, suggesting that targeting in-plant sources of contamination may be important for achieving low spore counts in the finished product. These data provide important insight regarding the diversity of spore populations isolated from dairy powders and bulk tank raw milk, and demonstrate that several spore genera are detected by multiple spore count methods. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Characterization of Clostridium difficile Spores Lacking Either SpoVAC or Dipicolinic Acid Synthetase

PubMed Central

Donnelly, M. Lauren; Fimlaid, Kelly A.

2016-01-01

ABSTRACT The spore-forming obligate anaerobe Clostridium difficile is a leading cause of antibiotic-associated diarrhea around the world. In order for C. difficile to cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. In C. difficile, cortex hydrolysis is necessary for DPA release, whereas in Bacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for C. difficile spore germination by constructing mutations in either spoVAC or dpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively. C. difficile spoVAC and dpaAB mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast, B. subtilis spoVAC and dpaAB mutant spores were unstable. Although C. difficile spoVAC and dpaAB mutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly, C. difficile spoVAC mutant spores were significantly more sensitive to heat treatment than dpaAB mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control C. difficile spore resistance and reveal differential requirements for these proteins among the Firmicutes. IMPORTANCE Clostridium difficile is a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential for C. difficile to cause disease, the factors required for this process have been only partially characterized. This study describes the roles of two factors, DpaAB and SpoVAC, which control the synthesis and release of dipicolinic acid (DPA), respectively, from bacterial spores. Previous studies of these proteins in other spore-forming organisms indicated that they are differentially required for spore formation, germination, and resistance. We now show that the proteins are dispensable for C. difficile spore formation and germination but are necessary for heat resistance. Thus, our study further highlights the diverse functions of DpaAB and SpoVAC in spore-forming organisms. PMID:27044622

A modified R-type bacteriocin specifically targeting Clostridium difficile prevents colonization of mice without affecting gut microbiota diversity.

PubMed

Gebhart, Dana; Lok, Stephen; Clare, Simon; Tomas, Myreen; Stares, Mark; Scholl, Dean; Donskey, Curtis J; Lawley, Trevor D; Govoni, Gregory R

2015-03-24

Clostridium difficile is a leading cause of nosocomial infections worldwide and has become an urgent public health threat requiring immediate attention. Epidemic lineages of the BI/NAP1/027 strain type have emerged and spread through health care systems across the globe over the past decade. Limiting person-to-person transmission and eradicating C. difficile, especially the BI/NAP1/027 strain type, from health care facilities are difficult due to the abundant shedding of spores that are impervious to most interventions. Effective prophylaxis for C. difficile infection (CDI) is lacking. We have genetically modified a contractile R-type bacteriocin ("diffocin") from C. difficile strain CD4 to kill BI/NAP1/027-type strains for this purpose. The natural receptor binding protein (RBP) responsible for diffocin targeting was replaced with a newly discovered RBP identified within a prophage of a BI/NAP1/027-type target strain by genome mining. The resulting modified diffocins (a.k.a. Avidocin-CDs), Av-CD291.1 and Av-CD291.2, were stable and killed all 16 tested BI/NAP1/027-type strains. Av-CD291.2 administered in drinking water survived passage through the mouse gastrointestinal (GI) tract, did not detectably alter the mouse gut microbiota or disrupt natural colonization resistance to C. difficile or the vancomycin-resistant Enterococcus faecium (VREF), and prevented antibiotic-induced colonization of mice inoculated with BI/NAP1/027-type spores. Given the high incidence and virulence of the pathogen, preventing colonization by BI/NAP1/027-type strains and limiting their transmission could significantly reduce the occurrence of the most severe CDIs. This modified diffocin represents a prototype of an Avidocin-CD platform capable of producing targetable, precision anti-C. difficile agents that can prevent and potentially treat CDIs without disrupting protective indigenous microbiota. Treatment and prevention strategies for bacterial diseases rely heavily on traditional antibiotics, which impose strong selection for resistance and disrupt protective microbiota. One consequence has been an upsurge of opportunistic pathogens, such as Clostridium difficile, that exploit antibiotic-induced disruptions in gut microbiota to proliferate and cause life-threatening diseases. We have developed alternative agents that utilize contractile bactericidal protein complexes (R-type bacteriocins) to kill specific C. difficile pathogens. Efficacy in a preclinical animal study indicates these molecules warrant further development as potential prophylactic agents to prevent C. difficile infections in humans. Since these agents do not detectably alter the indigenous gut microbiota or colonization resistance in mice, we believe they will be safe to administer as a prophylactic to block transmission in high-risk environments without rendering patients susceptible to enteric infection after cessation of treatment. Copyright © 2015 Gebhart et al.

Membrane filtration method for enumeration and isolation of Alicyclobacillus spp. from apple juice.

PubMed

Lee, S-Y; Chang, S-S; Shin, J-H; Kang, D-H

2007-11-01

To evaluate the applicability of filtration membranes for detecting Alicyclobacillus spp. spores in apple juice. Ten types of nitrocellulose membrane filters from five manufacturers were used to collect and enumerate five Alicyclobacillus spore isolates and results were compared to conventional K agar plating. Spore recovery differed among filters with an average recovery rate of 126.2%. Recovery levels also differed among spore isolates. Although significant difference (P < 0.05) in spore sizes existed, no correlation could be determined between spore size and membrane filter recovery rate. Recovery of spores using membrane filtration is dependent on the manufacturer and filter pore size. Correlations between spore recovery rate and spore size could not be determined. Low numbers of Alicyclobacillus spores in juice can be effectively detected using membrane filtration although recovery rate differences exist among different manufacturers. Use of membrane filtration is a simple, fast alternative to the week-long enrichment procedures currently employed in most quality assurance tests.

Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.

PubMed

Munoz, L; Sadaie, Y; Doi, R H

1978-10-10

The coat protein of Bacillus subtilis spores comprises about 10% of the total dry weight of spores and 25% of the total spore protein. One protein with a molecular weight of 13,000 to 15,000 comprises a major portion of the spore coat. This mature spore coat protein has histidine at its NH2 terminus and is relatively rich in hydrophobic amino acids. Netropsin, and antibiotic which binds to A-T-rich regions of DNA and inhibits sporulation, but not growth, decreased the synthesis of this spore coat protein by 75%. A precursor spore coat protein with a molecular weight of 25,000 is made initially at t1 of sporulation and is converted to the mature spore coat protein with a molecular weight of 13,500 at t2 - t3. These data indicate that the spore coat protein gene is expressed very early in sporulation prior to the modifications of RNA polymerase which have been noted.

Turbulent Dispersion of Pathogenic Spores Within and Above Plant Canopies: Field Experiments and Lagrangian Modeling

NASA Astrophysics Data System (ADS)

Gleicher, S.; Chamecki, M.; Isard, S.; Katul, G. G.

2012-12-01

Plant disease epidemics caused by pathogenic spores are a common and consequential threat to agricultural crops. In most cases, pathogenic spores are produced and released deep inside plant canopies and must be transported out of the canopy region in order to infect other fields and spread the disease. The fraction of spores that "escape" the canopy is crucial in determining how fast and far these plant diseases will spread. The goal of this work is to use a field experiment, coupled with a Lagrangian Stochastic Model (LSM), to investigate how properties of canopy turbulence impact the dispersion of spores inside the canopy and the fraction of spores that escape from the canopy. An extensive field experiment was conducted to study spore dispersion inside and outside a corn canopy. The spores were released from point sources located at various depths inside the canopy. Concentration measurements were obtained inside and above the canopy by a 3-dimensional grid of spore collectors. The experimental measurements of mean spore concentration are used to validate a LSM for spore dispersion. In the LSM, flow field statistics used to drive the particle dispersion are specified by a second-order closure model for turbulence within plant canopies. The dispersion model includes spore deposition on and rebound from canopy elements. The combination of experimental and numerical simulations is used to quantify the fraction of spores that escape the canopy. Effects of release height, friction velocity, and canopy architecture on the escape fraction of spores are explored using the LSM, and implications for disease propagation are discussed.

Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant, Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy

PubMed Central

Wang, Shiwei; Yu, Jing; Suvira, Milomir; Setlow, Peter; Li, Yong-qing

2015-01-01

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T1) and reached a maximum after the completion of CaDPA release (Trelease) and spore cortex lysis (Tlysis). PMID:26636757

Influence of Spore Moisture Content on the Dry-Heat Resistance of Bacillus subtilis var. niger

PubMed Central

Angelotti, Robert; Maryanski, James H.; Butler, Thomas F.; Peeler, James T.; Campbell, Jeptha E.

1968-01-01

The dry-heat resistance of Bacillus subtilis var. niger spores located in or on various materials was determined as D and z values in the range of 105 through 160 C. The systems tested included spores located on steel and paper strips, spores located between stainless-steel washers mated together under 150 inch-lb and 12 inch-lb of torque, and spores encapsulated in methylmethacrylate and epoxy plastics. D values for a given temperature varied with the test system. High D values were observed for the systems in which spores were encapsulated or under heavy torque, whereas lower D values were observed for the steel and paper strip systems and the lightly torqued system. Similar z values were obtained for the plastic and steel strip systems (zD = 21 C), but an unusually low z for spores on paper (zD = 12.9 C) and an unusually high z for spores on steel washers mated at 150 inch-lb of torque (zD = 32 C) were observed. The effect of spore moisture content on the D value of spores encapsulated in water-impermeable plastic was determined, and maximal resistance was observed for spores with a water activity (aw) of 0.2 to 0.4. Significantly decreased D values were observed for spores with moisture contents below aw 0.2 or above aw 0.4. The data indicate that the important factors to be considered when measuring the dry heat resistance of spores are (i) the initial moisture content of the spore, (ii) the rate of spore desiccation during heating, (iii) the water retention capacity of the material in or on which spores are located, and (iv) the relative humidity of the system at the test temperature. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 7 PMID:4968962

Genes That Bias Mendelian Segregation

PubMed Central

Grognet, Pierre; Lalucque, Hervé; Malagnac, Fabienne; Silar, Philippe

2014-01-01

Mendel laws of inheritance can be cheated by Meiotic Drive Elements (MDs), complex nuclear genetic loci found in various eukaryotic genomes and distorting segregation in their favor. Here, we identify and characterize in the model fungus Podospora anserina Spok1 and Spok2, two MDs known as Spore Killers. We show that they are related genes with both spore-killing distorter and spore-protecting responder activities carried out by the same allele. These alleles act as autonomous elements, exert their effects independently of their location in the genome and can act as MDs in other fungi. Additionally, Spok1 acts as a resistance factor to Spok2 killing. Genetical data and cytological analysis of Spok1 and Spok2 localization during the killing process suggest a complex mode of action for Spok proteins. Spok1 and Spok2 belong to a multigene family prevalent in the genomes of many ascomycetes. As they have no obvious cellular role, Spok1 and Spok2 Spore Killer genes represent a novel kind of selfish genetic elements prevalent in fungal genome that proliferate through meiotic distortion. PMID:24830502

Genes that bias Mendelian segregation.

PubMed

Grognet, Pierre; Lalucque, Hervé; Malagnac, Fabienne; Silar, Philippe

2014-01-01

Mendel laws of inheritance can be cheated by Meiotic Drive Elements (MDs), complex nuclear genetic loci found in various eukaryotic genomes and distorting segregation in their favor. Here, we identify and characterize in the model fungus Podospora anserina Spok1 and Spok2, two MDs known as Spore Killers. We show that they are related genes with both spore-killing distorter and spore-protecting responder activities carried out by the same allele. These alleles act as autonomous elements, exert their effects independently of their location in the genome and can act as MDs in other fungi. Additionally, Spok1 acts as a resistance factor to Spok2 killing. Genetical data and cytological analysis of Spok1 and Spok2 localization during the killing process suggest a complex mode of action for Spok proteins. Spok1 and Spok2 belong to a multigene family prevalent in the genomes of many ascomycetes. As they have no obvious cellular role, Spok1 and Spok2 Spore Killer genes represent a novel kind of selfish genetic elements prevalent in fungal genome that proliferate through meiotic distortion.

Metagenomic analysis of buffalo rumen microbiome: Effect of roughage diet on Dormancy and Sporulation genes.

PubMed

Singh, K M; Reddy, B; Patel, A K; Panchasara, H; Parmar, N; Patel, A B; Shah, T M; Bhatt, V D; Joshi, C G

2014-12-01

Buffalo rumen microbiome experiences a variety of diet stress and represents reservoir of Dormancy and Sporulation genes. However, the information on genomic responses to such conditions is very limited. The Ion Torrent PGM next generation sequencing technology was used to characterize general microbial diversity and the repertoire of microbial genes present, including genes associated with Dormancy and Sporulation in Mehsani buffalo rumen metagenome. The research findings revealed the abundance of bacteria at the domain level and presence of Dormancy and Sporulation genes which were predominantly associated with the Clostridia and Bacilli taxa belonging to the phyla Firmicutes. Genes associated with Sporulation cluster and Sporulation orphans were increased from 50% to 100% roughage treatment, thereby promoting sporulation all along the treatments. The spore germination is observed to be the highest in the 75% roughage treatment both in the liquid and solid rumen fraction samples with respect to the decrease in the values of the genes associated with spore core dehydration, thereby facilitating spore core hydration which is necessary for spore germination.

Manganese(II)-oxidizing Bacillus spores in Guaymas Basin hydrothermal sediments and plumes.

PubMed

Dick, Gregory J; Lee, Yifan E; Tebo, Bradley M

2006-05-01

Microbial oxidation and precipitation of manganese at deep-sea hydrothermal vents are important oceanic biogeochemical processes, yet nothing is known about the types of microorganisms or mechanisms involved. Here we report isolation of a number of diverse spore-forming Mn(II)-oxidizing Bacillus species from Guaymas Basin, a deep-sea hydrothermal vent environment in the Gulf of California, where rapid microbially mediated Mn(II) oxidation was previously observed. mnxG multicopper oxidase genes involved in Mn(II) oxidation were amplified from all Mn(II)-oxidizing Bacillus spores isolated, suggesting that a copper-mediated mechanism of Mn(II) oxidation could be important at deep-sea hydrothermal vents. Phylogenetic analysis of 16S rRNA and mnxG genes revealed that while many of the deep-sea Mn(II)-oxidizing Bacillus species are very closely related to previously recognized isolates from coastal sediments, other organisms represent novel strains and clusters. The growth and Mn(II) oxidation properties of these Bacillus species suggest that in hydrothermal sediments they are likely present as spores that are active in oxidizing Mn(II) as it emerges from the seafloor.

Analysis of selected fungi variation and its dependence on season and mountain range in southern Poland-key factors in drawing up trial guidelines for aeromycological monitoring.

PubMed

Pusz, Wojciech; Weber, Ryszard; Dancewicz, Andrzej; Kita, Włodzimierz

2017-09-27

The aim of the study was to identify fungal spores, in particular plant pathogenic fungi, occurring in the air in selected mountain ranges. The results revealed not only the array of fungal species migrating with air currents from the Czech Republic and Slovakia but also how the season of the year affects the distribution of spores. Such studies may lay a foundation for future aeromycological monitoring, in accordance with the requirements for integrated plant protection. Aeromycological research was carried out between 2013 and 2016 at 3-month intervals in mountainous areas along the southern borders of Poland: the Bieszczady, the Pieniny, the Giant Mountains (Karkonosze) and the Babia Góra Massif. The research relied on impact method employing Air Ideal 3P sampler, which, by drawing in atmospheric air, also collects fungal spores. Regardless of altitudinal zonation, the changing weather conditions appeared to be the main reason for the variations in the number of the fungal spores under study in those years.

Fungi in carpeting and furniture dust.

PubMed

Schober, G

1991-11-01

The qualitative and quantitative species composition of fungi in carpets and upholstered furniture dust found in the living-rooms of nine Dutch dwellings was examined in a pilot study. Numbers of spores of xerophilic fungi did not differ in dust removed from carpeting and upholstery. Spores of hydrophilic species were found to be more predominant on floors (P less than 0.05), whereas meso-hygrophilic spores, largely dominated by allergologically relevant Penicillium species, were significantly more abundant in dust taken from regularly used furniture (P less than 0.05). Our results indicate that growth conditions for fungi in the micro-habitats of furniture differ from those in carpeting. No statistically significant differences in number of viable spores have been found in samples taken from ground-floor level compared with those taken from 1st to 3rd floor level of dwellings. From this study, the need for a micro-topographic analysis of the fungal flora in the human environment has become apparent. Efficient allergological home sanitation in dwellings of allergic patients requires detailed data about the colonization of the various micro-habitats by allergenic fungi.

A molecular method to assess bioburden embedded within silicon-based resins used on modern spacecraft materials

NASA Astrophysics Data System (ADS)

Stam, Christina N.; Bruckner, James; Spry, J. Andy; Venkateswaran, Kasthuri; La Duc, Myron T.

2012-07-01

Current assessments of bioburden embedded in spacecraft materials are based on work performed in the Viking era (1970s), and the ability to culture organisms extracted from such materials. To circumvent the limitations of such approaches, DNA-based techniques were evaluated alongside established culturing techniques to determine the recovery and survival of bacterial spores encapsulated in spacecraft-qualified polymer materials. Varying concentrations of Bacillus pumilus SAFR-032 spores were completely embedded in silicone epoxy. An organic dimethylacetamide-based solvent was used to digest the epoxy and spore recovery was evaluated via gyrB-targeted qPCR, direct agar plating, most probably number analysis, and microscopy. Although full-strength solvent was shown to inhibit the germination and/or outgrowth of spores, dilution in excess of 100-fold allowed recovery with no significant decrease in cultivability. Similarly, qPCR (quantitative PCR) detection sensitivities as low as ~103 CFU ml-1 were achieved upon removal of inhibitory substances associated with the epoxy and/or solvent. These detection and enumeration methods show promise for use in assessing the embedded bioburden of spacecraft hardware.

Measuring Total and Germinable Spore Populations

NASA Technical Reports Server (NTRS)

Noell, A.C.; Yung, P.T.; Yang, W.; Lee, C.; Ponce, A.

2011-01-01

It has been shown that bacterial endospores can be enumerated using a microscopy based assay that images the luminescent halos from terbium ions bound to dipicolinic acid, a spore specific chemical marker released upon spore germination. Further development of the instrument has simplified it towards automation while at the same time improving image quality. Enumeration of total spore populations has also been developed allowing measurement of the percentage of viable spores in any population by comparing the germinable/culturable spores to the total. Percentage viability will allow a more quantitative comparison of the ability of spores to survive across a wide range of extreme environments.

Mushrooms use convectively created airflows to disperse their spores

PubMed Central

Dressaire, Emilie; Yamada, Lisa; Song, Boya; Roper, Marcus

2016-01-01

Thousands of basidiomycete fungal species rely on mushroom spores to spread across landscapes. It has long been thought that spores depend on favorable winds for dispersal—that active control of spore dispersal by the parent fungus is limited to an impulse delivered to the spores to carry them clear of the gill surface. Here we show that evaporative cooling of the air surrounding the pileus creates convective airflows capable of carrying spores at speeds of centimeters per second. Convective cells can transport spores from gaps that may be only 1 cm high and lift spores 10 cm or more into the air. This work reveals how mushrooms tolerate and even benefit from crowding and explains their high water needs. PMID:26929324

Selection of biological indicator for validating microwave heating sterilization.

PubMed

Sasaki, K; Mori, Y; Honda, W; Miyake, Y

1998-01-01

For the purpose of selecting an appropriate biological indicator for evaluation of the effects of microwave heating sterilization, we examined aerobic bacterial spores to determine whether microwaves have non-thermal sterilization effects. After microwave irradiation on dry bacterial spores (three species), none of the bacterial spores were killed. The survival rate of the spores after microwave irradiation of spore suspensions (twelve species) was compared with that after heating by a conventional method. The order of heat resistance in the bacterial species was similar between the two heating methods. Bacillus stearothermophilus spores were the most heat-resistant. These results suggest that microwaves have no non-thermal sterilization effects on bacterial spores, the specific resistant spores to microwave heating, and microwave heating sterilization can be evaluated in the same way as for conventional heating sterilization. As a biological indicator for evaluation of overkill sterilization, B. stearothermophilus spores may be appropriate for microwave heating sterilization as well as steam sterilization.

Comparison of hand hygiene procedures for removing Bacillus cereus spores.

PubMed

Sasahara, Teppei; Hayashi, Shunji; Hosoda, Kouichi; Morisawa, Yuji; Hirai, Yoshikazu

2014-01-01

Bacillus cereus is a spore-forming bacterium. B. cereus occasionally causes nosocomial infections, in which hand contamination with the spores plays an important role. Therefore, hand hygiene is the most important practice for controlling nosocomial B. cereus infections. This study aimed to determine the appropriate hand hygiene procedure for removing B. cereus spores. Thirty volunteers' hands were experimentally contaminated with B. cereus spores, after which they performed 6 different hand hygiene procedures. We compared the efficacy of the procedures in removing the spores from hands. The alcohol-based hand-rubbing procedures scarcely removed them. The soap washing procedures reduced the number of spores by more than 2 log10. Extending the washing time increased the spore-removing efficacy of the washing procedures. There was no significant difference in efficacy between the use of plain soap and antiseptic soap. Handwashing with soap is appropriate for removing B. cereus spores from hands. Alcohol-based hand-rubbing is not effective.

Efficacy of propidium iodide and FUN-1 stains for assessing viability in basidiospores of Rhizopogon roseolus.

PubMed

Fernández-Miranda, Elena; Majada, Juan; Casares, Abelardo

2017-01-01

The use of spores in applications of ectomycorrhizal fungi requires information regarding spore viability and germination, especially in genera such as Rhizopogon with high rates of spore dormancy. The authors developed a protocol to assess spore viability of Rhizopogon roseolus using four vital stains to quantify spore viability and germination and to optimize storage procedures. They showed that propidium iodide is an excellent stain for quantifying nonviable spores. Observing red fluorescent intravacuolar structures following staining with 2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide (FUN-1) can help identify viable spores that are activated. At 6 mo and 1 y, the spores kept in a water suspension survived better than those left within intact, dry gasterocarps. Our work highlights the importance of temperature, nutrients, and vitamins for maturation and germination of spores of R. roseolus during 1 y of storage.

Resistance to and killing by the sporicidal microbicide peracetic acid.

PubMed

Leggett, Mark J; Schwarz, J Spencer; Burke, Peter A; Mcdonnell, Gerald; Denyer, Stephen P; Maillard, Jean-Yves

2015-03-01

To elucidate the mechanisms of spore resistance to and killing by the oxidizing microbicide peracetic acid (PAA). Mutants of Bacillus subtilis lacking specific spore structures were used to identify resistance properties in spores and to understand the mechanism of action of PAA. We also assessed the effect of PAA treatment on a number of spore properties including heat tolerance, membrane integrity and germination. The spore coat is essential for spore PAA resistance as spores with defective coats were greatly sensitized to PAA treatment. Small acid-soluble spore proteins apparently provide no protection against PAA. Defects in spore germination, specifically in germination via the GerB and GerK but not the GerA germination receptors, as well as leakage of internal components suggest that PAA is active at the spore inner membrane. It is therefore likely that the inner membrane is the major site of PAA's sporicidal activity. PAA treatment targets the spore membrane, with some of its activity directed specifically against the GerB and GerK germination receptors. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Significance of air humidity and air velocity for fungal spore release into the air

NASA Astrophysics Data System (ADS)

Pasanen, A.-L.; Pasanen, P.; Jantunen, M. J.; Kalliokoski, P.

Our previous field studies have shown that the presence of molds in buildings does not necessarily mean elevated airborne spore counts. Therefore, we investigated the release of fungal spores from cultures of Aspergillus fumigatus, Penicillium sp. and Cladosporium sp. at different air velocities and air humidities. Spores of A. fumigatus and Penicillium sp. were released from conidiophores already at air velocity of 0.5 ms -1, whereas Cladosporium spores required at least a velocity of 1.0 ms -1. Airborne spore counts of A. fumigatus and Penicillium sp. were usually higher in dry than moist air, being minimal at relative humidities (r.h.) above 70%, while the effect of r.h. on the release of Cladosporium sp. was ambivalent. The geometric mean diameter of released spores increased when the r.h. exceeded a certain level which depends on fungal genus. Thus, spores of all three fungi were hygroscopic but the hygroscopicity of various spores appeared at different r.h.-ranges. This study indicates that spore release is controlled by external factors and depends on fungal genus which can be one reason for considerable variation of airborne spore counts in buildings with mold problems.

Development of an Aerosol Surface Inoculation Method for Bacillus Spores ▿

PubMed Central

Lee, Sang Don; Ryan, Shawn P.; Snyder, Emily Gibb

2011-01-01

A method was developed to deposit Bacillus subtilis spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with Bacillus subtilis spores, a surrogate for Bacillus anthracis. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 107 CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies. PMID:21193670

Development of an aerosol surface inoculation method for bacillus spores.

PubMed

Lee, Sang Don; Ryan, Shawn P; Snyder, Emily Gibb

2011-03-01

A method was developed to deposit Bacillus subtilis spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with Bacillus subtilis spores, a surrogate for Bacillus anthracis. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 10(7) CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies.

PpASCL, the Physcomitrella patens Anther-Specific Chalcone Synthase-Like Enzyme Implicated in Sporopollenin Biosynthesis, Is Needed for Integrity of the Moss Spore Wall and Spore Viability

PubMed Central

Daku, Rhys M.; Rabbi, Fazle; Buttigieg, Josef; Coulson, Ian M.; Horne, Derrick; Martens, Garnet; Ashton, Neil W.; Suh, Dae-Yeon

2016-01-01

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores. PMID:26752629

Monitoring the Wet-Heat Inactivation Dynamics of Single Spores of Bacillus Species by Using Raman Tweezers, Differential Interference Contrast Microscopy, and Nucleic Acid Dye Fluorescence Microscopy▿

PubMed Central

Zhang, Pengfei; Kong, Lingbo; Wang, Guiwen; Setlow, Peter; Li, Yong-qing

2011-01-01

Dynamic processes during wet-heat treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis at 80 to 90°C were investigated using dual-trap Raman spectroscopy, differential interference contrast (DIC) microscopy, and nucleic acid stain (SYTO 16) fluorescence microscopy. During spore wet-heat treatment, while the spores' 1:1 chelate of Ca2+ with dipicolinic acid (CaDPA) was released rapidly at a highly variable time Tlag, the levels of spore nucleic acids remained nearly unchanged, and the Tlag times for individual spores from the same preparation were increased somewhat as spore levels of CaDPA increased. The brightness of the spores' DIC image decreased by ∼50% in parallel with CaDPA release, and there was no spore cortex hydrolysis observed. The lateral diameters of the spores' DIC image and SYTO 16 fluorescence image also decreased in parallel with CaDPA release. The SYTO 16 fluorescence intensity began to increase during wet-heat treatment at a time before Tlag and reached maximum at a time slightly later than Trelease. However, the fluorescence intensities of wet-heat-inactivated spores were ∼15-fold lower than those of nutrient-germinated spores, and this low SYTO 16 fluorescence intensity may be due in part to the low permeability of the dormant spores' inner membranes to SYTO 16 and in part to nucleic acid denaturation during the wet-heat treatment. PMID:21602365

Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA2mob Operon in Foodborne Strains of Bacillus subtilis.

PubMed

Krawczyk, Antonina O; de Jong, Anne; Omony, Jimmy; Holsappel, Siger; Wells-Bennik, Marjon H J; Kuipers, Oscar P; Eijlander, Robyn T

2017-04-01

Spore heat resistance, germination, and outgrowth are problematic bacterial properties compromising food safety and quality. Large interstrain variation in these properties makes prediction and control of spore behavior challenging. High-level heat resistance and slow germination of spores of some natural Bacillus subtilis isolates, encountered in foods, have been attributed to the occurrence of the spoVA 2mob operon carried on the Tn 1546 transposon. In this study, we further investigate the correlation between the presence of this operon in high-level-heat-resistant spores and their germination efficiencies before and after exposure to various sublethal heat treatments (heat activation, or HA), which are known to significantly improve spore responses to nutrient germinants. We show that high-level-heat-resistant spores harboring spoVA 2mob required higher HA temperatures for efficient germination than spores lacking spoVA 2mob The optimal spore HA requirements additionally depended on the nutrients used to trigger germination, l-alanine (l-Ala), or a mixture of l-asparagine, d-glucose, d-fructose, and K + (AGFK). The distinct HA requirements of these two spore germination pathways are likely related to differences in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved in this germination response. In contrast, no relation was found between transcription levels of main germination genes and spore germination phenotypes. The findings presented in this study have great implications for practices in the food industry, where heat treatments are commonly used to inactivate pathogenic and spoilage microbes, including bacterial spore formers. IMPORTANCE This study describes a strong variation in spore germination capacities and requirements for a heat activation treatment, i.e., an exposure to sublethal heat that increases spore responsiveness to nutrient germination triggers, among 17 strains of B. subtilis , including 9 isolates from spoiled food products. Spores of industrial foodborne isolates exhibited, on average, less efficient and slower germination responses and required more severe heat activation than spores from other sources. High heat activation requirements and inefficient, slow germination correlated with elevated resistance of spores to heat and with specific genetic features, indicating a common genetic basis of these three phenotypic traits. Clearly, interstrain variation and numerous factors that shape spore germination behavior challenge standardization of methods to recover highly heat-resistant spores from the environment and have an impact on the efficacy of preservation techniques used by the food industry to control spores. Copyright © 2017 American Society for Microbiology.

Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA2mob Operon in Foodborne Strains of Bacillus subtilis

PubMed Central

Krawczyk, Antonina O.; de Jong, Anne; Omony, Jimmy; Holsappel, Siger; Wells-Bennik, Marjon H. J.; Eijlander, Robyn T.

2017-01-01

ABSTRACT Spore heat resistance, germination, and outgrowth are problematic bacterial properties compromising food safety and quality. Large interstrain variation in these properties makes prediction and control of spore behavior challenging. High-level heat resistance and slow germination of spores of some natural Bacillus subtilis isolates, encountered in foods, have been attributed to the occurrence of the spoVA2mob operon carried on the Tn1546 transposon. In this study, we further investigate the correlation between the presence of this operon in high-level-heat-resistant spores and their germination efficiencies before and after exposure to various sublethal heat treatments (heat activation, or HA), which are known to significantly improve spore responses to nutrient germinants. We show that high-level-heat-resistant spores harboring spoVA2mob required higher HA temperatures for efficient germination than spores lacking spoVA2mob. The optimal spore HA requirements additionally depended on the nutrients used to trigger germination, l-alanine (l-Ala), or a mixture of l-asparagine, d-glucose, d-fructose, and K+ (AGFK). The distinct HA requirements of these two spore germination pathways are likely related to differences in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved in this germination response. In contrast, no relation was found between transcription levels of main germination genes and spore germination phenotypes. The findings presented in this study have great implications for practices in the food industry, where heat treatments are commonly used to inactivate pathogenic and spoilage microbes, including bacterial spore formers. IMPORTANCE This study describes a strong variation in spore germination capacities and requirements for a heat activation treatment, i.e., an exposure to sublethal heat that increases spore responsiveness to nutrient germination triggers, among 17 strains of B. subtilis, including 9 isolates from spoiled food products. Spores of industrial foodborne isolates exhibited, on average, less efficient and slower germination responses and required more severe heat activation than spores from other sources. High heat activation requirements and inefficient, slow germination correlated with elevated resistance of spores to heat and with specific genetic features, indicating a common genetic basis of these three phenotypic traits. Clearly, interstrain variation and numerous factors that shape spore germination behavior challenge standardization of methods to recover highly heat-resistant spores from the environment and have an impact on the efficacy of preservation techniques used by the food industry to control spores. PMID:28130296

Bacillus Endospores - an ideal exobiological Tool

NASA Astrophysics Data System (ADS)

Moeller, R.; Horneck, G.

Exobiology investigations have one overall goal -- finding the answer to the question if Earth is the only possible place in our universe where life was created. For tackling this question a good approach is to use a simple and ubiquitous system like bacteria as used in BIOPAN and EXPOSE. Many of these microorganisms have the ability to form metabolic inactive continuous forms such as Bacillus endospores. These spores are highly resistant against a variety of environmental stresses, such as toxic chemical agents, desiccation, high and low pressure, high doses of ionising and UV radiation and temperature extremes such as heat or permafrost. They are ubiquitous, inhabit soils and rocks and are easily disseminated by wind and water. Therefore they are suitable test systems for studying several questions of astrobiology, such as the theory of Panspermia, planetary protection issues in connection with missions to Mars or Europa, or chances for life on past or present Mars. The strategies Bacillus sp. endospores have developed to survive harsh conditions include a desiccated spore core, an altered conformation of their DNA (A-form), high concentration of small acid-soluble proteins (SASPs) stabilising the DNA, dipicolinic acid (DPA) for stabilisation and protective spore coating layers. We have investigated the role of endogenous and exogenous pigments in the UV-resistance of Bacillus endospores by using spores of different degree and kind of pigmentation, i.e. white, grey or red spores (DSMZ culture collection). The spectral ranges of UV radiation represented those of the early or present UV radiation climate of Earth or Mars. It was found, that endogenous carotenoids, identified by spectrophotometrical analysis from a spore extract as well as in-situ by Raman spectroscopy, efficiently protect against UV-A radiation, whereas melanin was also protective against UV-C radiation. From these studied follows, that highly pigmented spores might survive even in an intense UV radiation climate, such as that on Mars or early Earth.

Evaluation of some physical and chemical treatments for inactivating microsporidian spores isolated from fish.

PubMed

Leiro, José M; Piazzon, Carla; Domínguez, Berta; Mallo, Natalia; Lamas, Jesús

2012-05-15

Microsporidia are a large diverse group of intracellular parasites now considered as fungi. They are particularly prevalent in fish and are recognized as important opportunistic parasites in humans. Although the mode of transmission of microsporidia has not been fully clarified, the consumption and manipulation of infected fish may be a risk factor for humans. Comparative analysis of rDNA sequence revealed that the microsporidians used in the present study had 99-100% identity with anglerfish microsporidians of the genus Spraguea and very low identity with microsporidians that infect humans. Microsporidian spores were exposed to different physical and chemical treatments: freezing at -20°C for 24-78 h, heating at 60°C for 5-15 min, microwaving at 700 W, 2.45 GHz for 15-60s, and treatment with ethanol at concentrations of between 1 and 70% for 15 min. The viability of the spores after each treatment was evaluated by two methods: a) haemocytometer counts, measuring the extrusion of the polar filament in control and treated spores, and b) a fluorometric method, testing the membrane integrity by propidium iodide exclusion. The results of both methods were concordant. Spores were inactivated by freezing at -20°C for more than 48 h, by heating to 60°C for 10 min and by microwaving at 750 W, for 20s. Exposure to 70% ethanol for 15 min also inactivated microsporidian spores. The results suggest that both freezing and heating are effective treatments for destroying microsporidian spores in European white anglerfish, and that 70% ethanol could be used by fish processors to disinfect their hands and the utensils used in processing fish. The fluorometric method can be used as an alternative to haemocytometer counts in disinfection studies aimed at establishing strategies for inactivating and reducing the viability and the potential infectivity of microsporidians present in fish or in the environment. Copyright © 2012 Elsevier B.V. All rights reserved.

Stratosphere Conditions Inactivate Bacterial Endospores from a Mars Spacecraft Assembly Facility.

PubMed

Khodadad, Christina L; Wong, Gregory M; James, Leandro M; Thakrar, Prital J; Lane, Michael A; Catechis, John A; Smith, David J

2017-04-01

Every spacecraft sent to Mars is allowed to land viable microbial bioburden, including hardy endospore-forming bacteria resistant to environmental extremes. Earth's stratosphere is severely cold, dry, irradiated, and oligotrophic; it can be used as a stand-in location for predicting how stowaway microbes might respond to the martian surface. We launched E-MIST, a high-altitude NASA balloon payload on 10 October 2015 carrying known quantities of viable Bacillus pumilus SAFR-032 (4.07 × 10 7 spores per sample), a radiation-tolerant strain collected from a spacecraft assembly facility. The payload spent 8 h at ∼31 km above sea level, exposing bacterial spores to the stratosphere. We found that within 120 and 240 min, spore viability was significantly reduced by 2 and 4 orders of magnitude, respectively. By 480 min, <0.001% of spores carried to the stratosphere remained viable. Our balloon flight results predict that most terrestrial bacteria would be inactivated within the first sol on Mars if contaminated spacecraft surfaces receive direct sunlight. Unfortunately, an instrument malfunction prevented the acquisition of UV light measurements during our balloon mission. To make up for the absence of radiometer data, we calculated a stratosphere UV model and conducted ground tests with a 271.1 nm UVC light source (0.5 W/m 2 ), observing a similarly rapid inactivation rate when using a lower number of contaminants (640 spores per sample). The starting concentration of spores and microconfiguration on hardware surfaces appeared to influence survivability outcomes in both experiments. With the relatively few spores that survived the stratosphere, we performed a resequencing analysis and identified three single nucleotide polymorphisms compared to unexposed controls. It is therefore plausible that bacteria enduring radiation-rich environments (e.g., Earth's upper atmosphere, interplanetary space, or the surface of Mars) may be pushed in evolutionarily consequential directions. Key Words: Planetary protection-Stratosphere-Balloon-Mars analog environment-E-MIST payload-Bacillus pumilus SAFR-032. Astrobiology 17, 337-350.

Stratosphere Conditions Inactivate Bacterial Endospores from a Mars Spacecraft Assembly Facility

NASA Astrophysics Data System (ADS)

Khodadad, Christina L.; Wong, Gregory M.; James, Leandro M.; Thakrar, Prital J.; Lane, Michael A.; Catechis, John A.; Smith, David J.

2017-04-01

Every spacecraft sent to Mars is allowed to land viable microbial bioburden, including hardy endospore-forming bacteria resistant to environmental extremes. Earth's stratosphere is severely cold, dry, irradiated, and oligotrophic; it can be used as a stand-in location for predicting how stowaway microbes might respond to the martian surface. We launched E-MIST, a high-altitude NASA balloon payload on 10 October 2015 carrying known quantities of viable Bacillus pumilus SAFR-032 (4.07 × 107 spores per sample), a radiation-tolerant strain collected from a spacecraft assembly facility. The payload spent 8 h at ˜31 km above sea level, exposing bacterial spores to the stratosphere. We found that within 120 and 240 min, spore viability was significantly reduced by 2 and 4 orders of magnitude, respectively. By 480 min, <0.001% of spores carried to the stratosphere remained viable. Our balloon flight results predict that most terrestrial bacteria would be inactivated within the first sol on Mars if contaminated spacecraft surfaces receive direct sunlight. Unfortunately, an instrument malfunction prevented the acquisition of UV light measurements during our balloon mission. To make up for the absence of radiometer data, we calculated a stratosphere UV model and conducted ground tests with a 271.1 nm UVC light source (0.5 W/m2), observing a similarly rapid inactivation rate when using a lower number of contaminants (640 spores per sample). The starting concentration of spores and microconfiguration on hardware surfaces appeared to influence survivability outcomes in both experiments. With the relatively few spores that survived the stratosphere, we performed a resequencing analysis and identified three single nucleotide polymorphisms compared to unexposed controls. It is therefore plausible that bacteria enduring radiation-rich environments (e.g., Earth's upper atmosphere, interplanetary space, or the surface of Mars) may be pushed in evolutionarily consequential directions.

Evaluation of fungal spore characteristics in Beijing, China, based on molecular tracer measurements

NASA Astrophysics Data System (ADS)

Liang, Linlin; Engling, Guenter; He, Kebin; Du, Zhenyu; Cheng, Yuan; Duan, Fengkui

2013-03-01

PM2.5 (particulate matter with aerodynamic diameters less than 2.5 μm) and PM10 (particulate matter with aerodynamic diameters less than 10 μm) samples were collected by high-volume air samplers simultaneously at a rural site and an urban site in Beijing, China. Various carbohydrates were quantified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), including the sugar alcohols mannitol and arabitol, recently proposed as molecular tracers for fungal aerosol. The annual average concentrations of arabitol in PM2.5 and PM10 at the urban site were 7.4 ± 9.4 and 21.0 ± 20.4 ng m-3, and the respective mannitol concentrations were 10.3 ± 9.5 and 31.9 ± 26.9 ng m-3. During summer and autumn, higher arabitol and mannitol levels than during spring and winter were observed in coarse particles, probably due to different dominant sources of fungal spores in different seasons. In the dry season (i.e., winter and spring) in Beijing, probably only the suspension from exposed surfaces (e.g., soil resuspension, transported dust, etc) can be regarded as the main sources for fungal aerosols. On the other hand, in summer and autumn, fungal spores in the atmosphere can be derived from more complex sources, including plants, vegetation decomposition and agricultural activity, such as ploughing; these fungal spore sources may contribute more to coarse PM. Moreover, statistical analysis according to typical seasonal patterns, including a dry season (December 2010 to March 2011) and a wet season (July to September 2011), revealed different variations of fungal spores in different seasons. Although fungal spore levels at rural sites were reported to be consistently higher than those at urban sites in other studies, our findings showed the opposite pattern, indicating a high abundance of fungal spores in the urban area of this Chinese megacity.

Allelic differences within and among sister spores of the arbuscular mycorrhizal fungus Glomus etunicatum suggest segregation at sporulation.

PubMed

Boon, Eva; Zimmerman, Erin; St-Arnaud, Marc; Hijri, Mohamed

2013-01-01

Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi that form mutualistic symbioses with their host plants. AMF are made up of coenocytic networks of hyphae through which nuclei and organelles can freely migrate. In this study, we investigated the possibility of a genetic bottleneck and segregation of allelic variation at sporulation for a low-copy Polymerase1-like gene, PLS. Specifically, our objectives were (1) to estimate what allelic diversity is passed on to a single spore (2) to determine whether this diversity is less than the total amount of variation found in all spores (3) to investigate whether there is any differential segregation of allelic variation. We inoculated three tomato plants with a single spore of Glomus etunicatum each and after six months sampled between two and three daughter spores per tomato plant. Pyrosequencing PLS amplicons in eight spores revealed high levels of allelic diversity; between 43 and 152 alleles per spore. We corroborated the spore pyrosequencing results with Sanger- and pyrosequenced allele distributions from the original parent isolate. Both sequencing methods retrieved the most abundant alleles from the offspring spore allele distributions. Our results indicate that individual spores contain only a subset of the total allelic variation from the pooled spores and parent isolate. Patterns of allele diversity between spores suggest the possibility for segregation of PLS alleles among spores. We conclude that a genetic bottleneck could potentially occur during sporulation in AMF, with resulting differences in genetic variation among sister spores. We suggest that the effects of this bottleneck may be countered by anastomosis (hyphal fusion) between related hyphae.

The cellulose-binding activity of the PsB multiprotein complex is required for proper assembly of the spore coat and spore viability in Dictyostelium discoideum.

PubMed

Srinivasan, S; Griffiths, K R; McGuire, V; Champion, A; Williams, K L; Alexander, S

2000-08-01

The terminal event of spore differentiation in the cellular slime mould Dictyostelium discoideum is the assembly of the spore coat, which surrounds the dormant amoeba and allows the organism to survive during extended periods of environmental stress. The spore coat is a polarized extracellular matrix composed of glycoproteins and cellulose. The process of spore coat formation begins by the regulated secretion of spore coat proteins from the prespore vesicles (PSVs). Four of the major spore coat proteins (SP96, PsB/SP85, SP70 and SP60) exist as a preassembled multiprotein complex within the PSVs. This complete complex has an endogenous cellulose-binding activity. Mutant strains lacking either the SP96 or SP70 proteins produce partial complexes that do not have cellulose-binding activity, while mutants lacking SP60 produce a partial complex that retains this activity. Using a combination of immunofluorescence microscopy and biochemical methods we now show that the lack of cellulose-binding activity in the SP96 and SP70 mutants results in abnormally assembled spore coats and spores with greatly reduced viability. In contrast, the SP60 mutant, in which the PsB complex retains its cellulose-binding activity, produces spores with apparently unaltered structure and viability. Thus, it is the loss of the cellulose-binding activity of the PsB complex, rather than the mere loss of individual spore coat proteins, that results in compromised spore coat structure. These results support the idea that the cellulose-binding activity associated with the complete PsB complex plays an active role in the assembly of the spore coat.

Carvacrol suppresses high pressure high temperature inactivation of Bacillus cereus spores.

PubMed

Luu-Thi, Hue; Corthouts, Jorinde; Passaris, Ioannis; Grauwet, Tara; Aertsen, Abram; Hendrickx, Marc; Michiels, Chris W

2015-03-16

The inactivation of bacterial spores generally proceeds faster and at lower temperatures when heat treatments are conducted under high pressure, and high pressure high temperature (HPHT) processing is, therefore, receiving an increased interest from food processors. However, the mechanisms of spore inactivation by HPHT treatment are poorly understood, particularly at moderately elevated temperature. In the current work, we studied inactivation of the spores of Bacillus cereus F4430/73 by HPHT treatment for 5 min at 600MPa in the temperature range of 50-100°C, using temperature increments of 5°C. Additionally, we investigated the effect of the natural antimicrobial carvacrol on spore germination and inactivation under these conditions. Spore inactivation by HPHT was less than about 1 log unit at 50 to 70°C, but gradually increased at higher temperatures up to about 5 log units at 100°C. DPA release and loss of spore refractility in the spore population were higher at moderate (≤65°C) than at high (≥70°C) treatment temperatures, and we propose that moderate conditions induced the normal physiological pathway of spore germination resulting in fully hydrated spores, while at higher temperatures this pathway was suppressed and replaced by another mechanism of pressure-induced dipicolinic acid (DPA) release that results only in partial spore rehydration, probably because spore cortex hydrolysis is inhibited. Carvacrol strongly suppressed DPA release and spore rehydration during HPHT treatment at ≤65°C and also partly inhibited DPA release at ≥65°C. Concomitantly, HPHT spore inactivation was reduced by carvacrol at 65-90°C but unaffected at 95-100°C. Copyright © 2014 Elsevier B.V. All rights reserved.

Allelic Differences within and among Sister Spores of the Arbuscular Mycorrhizal Fungus Glomus etunicatum Suggest Segregation at Sporulation

PubMed Central

St-Arnaud, Marc; Hijri, Mohamed

2013-01-01

Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi that form mutualistic symbioses with their host plants. AMF are made up of coenocytic networks of hyphae through which nuclei and organelles can freely migrate. In this study, we investigated the possibility of a genetic bottleneck and segregation of allelic variation at sporulation for a low-copy Polymerase1-like gene, PLS. Specifically, our objectives were (1) to estimate what allelic diversity is passed on to a single spore (2) to determine whether this diversity is less than the total amount of variation found in all spores (3) to investigate whether there is any differential segregation of allelic variation. We inoculated three tomato plants with a single spore of Glomus etunicatum each and after six months sampled between two and three daughter spores per tomato plant. Pyrosequencing PLS amplicons in eight spores revealed high levels of allelic diversity; between 43 and 152 alleles per spore. We corroborated the spore pyrosequencing results with Sanger- and pyrosequenced allele distributions from the original parent isolate. Both sequencing methods retrieved the most abundant alleles from the offspring spore allele distributions. Our results indicate that individual spores contain only a subset of the total allelic variation from the pooled spores and parent isolate. Patterns of allele diversity between spores suggest the possibility for segregation of PLS alleles among spores. We conclude that a genetic bottleneck could potentially occur during sporulation in AMF, with resulting differences in genetic variation among sister spores. We suggest that the effects of this bottleneck may be countered by anastomosis (hyphal fusion) between related hyphae. PMID:24386173

Role of YpeB in Cortex Hydrolysis during Germination of Bacillus anthracis Spores

PubMed Central

Bernhards, Casey B.

2014-01-01

The infectious agent of the disease anthrax is the spore of Bacillus anthracis. Bacterial spores are extremely resistant to environmental stresses, which greatly hinders spore decontamination efforts. The spore cortex, a thick layer of modified peptidoglycan, contributes to spore dormancy and resistance by maintaining the low water content of the spore core. The cortex is degraded by germination-specific lytic enzymes (GSLEs) during spore germination, rendering the cells vulnerable to common disinfection techniques. This study investigates the relationship between SleB, a GSLE in B. anthracis, and YpeB, a protein necessary for SleB stability and function. The results indicate that ΔsleB and ΔypeB spores exhibit similar germination phenotypes and that the two proteins have a strict codependency for their incorporation into the dormant spore. In the absence of its partner protein, SleB or YpeB is proteolytically degraded soon after expression during sporulation, rather than escaping the developing spore. The three PepSY domains of YpeB were examined for their roles in the interaction with SleB. YpeB truncation mutants illustrate the necessity of a region beyond the first PepSY domain for SleB stability. Furthermore, site-directed mutagenesis of highly conserved residues within the PepSY domains resulted in germination defects corresponding to reduced levels of both SleB and YpeB in the mutant spores. These results identify residues involved in the stability of both proteins and reiterate their codependent relationship. It is hoped that the study of GSLEs and interacting proteins will lead to the use of GSLEs as targets for efficient activation of spore germination and facilitation of spore cleanup. PMID:25022853

Use of Magnetic Bead Resin and Automated Liquid Handler Extraction Methods to Robotically Isolate Nucleic Acids of Biological Agent Simulates

DTIC Science & Technology

2003-09-01

concentration, and Bacillus subtilis var. niger spores were detectable at 10,000 CFU/ml. When combined with bead beating, these spores were consistently...Bioloeical Aaent Simulants. Cell suspensions of Bacillus subtilis var. niger spores (BG spores ) and Erwinia herbicola vegetative cells were prepared for...use as biological simulants. BG spores were prepared by inoculating 1 g spores of Bacillus subtilis var. niger (Merck & Co., Inc., Whitehouse Station

Walking dead: Permeabilization of heat-treated Geobacillus stearothermophilus ATCC 12980 spores under growth-preventing conditions.

PubMed

Mtimet, Narjes; Trunet, Clément; Mathot, Anne-Gabrielle; Venaille, Laurent; Leguérinel, Ivan; Coroller, Louis; Couvert, Olivier

2017-06-01

Although heat treatment is probably the oldest and the most common method used to inactivate spores in food processes, the specific mechanism of heat killing of spores is still not fully understood. The purpose of this study is to investigate the evolution of the permeabilization and the viability of heat-treated spores during storage under growth-preventing conditions. Geobacillus stearothermophilus spores were heat-treated under various conditions of temperature and pH, and then stored under conditions of temperature and pH that prevent growth. Spore survival was evaluated by count plating immediately after heat treatment, and then during storage over a period of months. Flow cytometry analyses were performed to investigate the Syto 9 permeability of heat-treated spores. Sub-lethally heat-treated spores of G. stearothermophilus were physically committed to permeabilization after heat treatment. However, prolonged heat treatment may abolish the spore permeabilization and block heat-treated spores in the refractive state. However, viability loss and permeabilization during heat treatment seem to be two different mechanisms that occur independently, and the loss of permeabilization properties takes place at a much slower rate than spore killing. Under growth-preventing conditions, viable heat-treated spores presumably lose their viability due to the permeabilization phenomena, which makes them more susceptible to the action of adverse conditions precluding growth. Copyright © 2016 Elsevier Ltd. All rights reserved.

Arbuscular mycorrhizal fungi in Mimosa tenuiflora (Willd.) Poir from Brazilian semi-arid.

PubMed

de Souza, Tancredo Augusto Feitosa; Rodriguez-Echeverría, Susana; de Andrade, Leonaldo Alves; Freitas, Helena

2016-01-01

Many plant species from Brazilian semi-arid present arbuscular mycorrhizal fungi (AMF) in their rhizosphere. These microorganisms play a key role in the establishment, growth, survival of plants and protection against drought, pathogenic fungi and nematodes. This study presents a quantitative analysis of the AMF species associated with Mimosa tenuiflora, an important native plant of the Caatinga flora. AMF diversity, spore abundance and root colonization were estimated in seven sampling locations in the Ceará and Paraíba States, during September of 2012. There were significant differences in soil properties, spore abundance, percentage of root colonization, and AMF diversity among sites. Altogether, 18 AMF species were identified, and spores of the genera Acaulospora, Claroideoglomus, Dentiscutata, Entrophospora, Funneliformis, Gigaspora, Glomus, Racocetra, Rhizoglomus and Scutellospora were observed. AMF species diversity and their spore abundance found in M. tenuiflora rhizosphere shown that this native plant species is an important host plant to AMF communities from Brazilian semi-arid region. We concluded that: (a) during the dry period and in semi-arid conditions, there is a high spore production in M. tenuiflora root zone; and (b) soil properties, as soil pH and available phosphorous, affect AMF species diversity, thus constituting key factors for the similarity/dissimilarity of AMF communities in the M. tenuiflora root zone among sites. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

The orphan germinant receptor protein GerXAO (but not GerX3b) is essential for L-alanine induced germination in Clostridium botulinum Group II.

PubMed

Brunt, Jason; Carter, Andrew T; Pye, Hannah V; Peck, Michael W

2018-05-04

Clostridium botulinum is an anaerobic spore forming bacterium that produces the potent botulinum neurotoxin that causes a severe and fatal neuro-paralytic disease of humans and animals (botulism). C. botulinum Group II is a psychrotrophic saccharolytic bacterium that forms spores of moderate heat resistance and is a particular hazard in minimally heated chilled foods. Spore germination is a fundamental process that allows the spore to transition to a vegetative cell and typically involves a germinant receptor (GR) that responds to environmental signals. Analysis of C. botulinum Group II genomes shows they contain a single GR cluster (gerX3b), and an additional single gerA subunit (gerXAO). Spores of C. botulinum Group II strain Eklund 17B germinated in response to the addition of L-alanine, but did not germinate following the addition of exogenous Ca 2+ -DPA. Insertional inactivation experiments in this strain unexpectedly revealed that the orphan GR GerXAO is essential for L-alanine stimulated germination. GerX3bA and GerX3bC affected the germination rate but were unable to induce germination in the absence of GerXAO. No role could be identified for GerX3bB. This is the first study to identify the functional germination receptor of C. botulinum Group II.

In vitro production of Clostridium difficile spores for use in the efficacy evaluation of disinfectants: a precollaborative investigation.

PubMed

Hasan, Jafrul A; Japal, Knoxley M; Christensen, Erick R; Samalot-Freire, Luisa C

2011-01-01

Clostridium difficile is a strict anaerobic spore-forming bacterium, and an increasingly common nosocomial pathogen. The U.S. Environmental Protection Agency (EPA) is responsible for the registration of disinfectants, including products designed to treat environmental surfaces contaminated with spores of C. difficile. Product efficacy data are required for registration; however, there is a lack of methodology for generating high-quality spore suspensions for evaluating product performance. As such, a study was carried out to select a suitable C. difficile strain and to develop a stand-alone method to prepare a spore suspension that meets specific criteria necessary for quantitative testing of disinfectants. The criteria are: (1) a spore titer of > 8 log10/mL, (2) > or = 90% spores to vegetative cells, and (3) resistance of spores (determined by viability) to 2.5 M hydrochloric acid (HCl). Several strains of C. difficile (toxigenic and nontoxigenic) were grown on various media (solid and liquid) for varying lengths of time to determine the best combination of incubation conditions and media to optimize spore production and quality. Once the spore production procedure was optimized, a toxigenic strain of C. difficile [American Type Culture Collection (ATCC) 43598] was selected for use in trials to verify repeatability from one production run to the next. The spore suspension was initiated by spreading vegetative cells of C. difficile (ATCC 43598) on CDC anaerobic 5% sheep blood agar plates and incubating for 7-10 days at 36 +/- 1 degrees C under anaerobic conditions. Spores were harvested when > or = 90% of the cells converted to spores as determined by observation using phase-contrast microscopy. The spores were washed three times with saline-Tween-80, resuspended in cold deionized water, heated to 70 degrees C for 10 min, evaluated microscopically for quality, and enumerated on cycloserine-cefoxitin-fructose agar containing horse blood and taurocholate. The spore suspension was used to inoculate brushed stainless steel carriers (1 cm in diameter) with and without a soil load in accordance with the Standard Quantitative Carrier Disk Test Method (ASTM E-2197-02) to determine carrier load. Once it was determined that > 6 log10 spores/carrier could be recovered, spores were evaluated for resistance to HCI. The sporulation method presented in this report is simple and repeatable and results in spore suspension of high titer (> 8 log10/mL) and quality (> or = 90% spores to vegetative cells) that met acid resistance criteria (spores were resistant to 2.5 M HCI for 10 min). In addition, recovery from brushed stainless steel carriers with and without soil load was > 6 log10 spores/carrier. A 6 log10 performance standard was set forth in the EPA's interim guidance for generating data to support a label claim for effectiveness against C. difficile spores on hard, nonporous surfaces. This precollaborative investigation successfully demonstrated the use of a methodology for in vitro production of C. difficile spores (ATCC 43598) necessary for conducting efficacy tests. A proposal will be submitted to the AOAC INTERNATIONAL Methods Committee on Antimicrobial Efficacy Testing for a collaborative study; see Appendix.

[Sporocidic activity of sodium hypochlorite and peracetic acid alone or combined against free or fixed spores or on biofilm].

PubMed

Samrakandi, M M; Roques, C; Michel, G

1994-05-01

In order to assess the sporocidal activity of chlorine and peracetic acid (PAA), alone and in combination, against a spored biofilm, the biofilms of two species (Bacillus subtilis ATCC 6633 and Bacillus megaterium ATCC 8245) were formed on inert support (tygon). A sporulation kinetic of these bacteria in biofilm was established. Sporocidal properties of chlorine and PAA were compared against free spores, spores fixed by drying and spores in biofilm. The combination of these two products was also tested. Minimal sporocidal concentrations (MSC) of the two products towards free spores were determined (contact time 5 mn). The efficacy of these MSC were evaluated in terms of contact time on adhered spores and on spores in biofilm. Chlorine and PAA exhibited an excellent sporocidal activity. The combination of PAA and chlorine, tested by checkerboard micromethod, was found to be synergistic in case of free or adhered spores. The spored biofilm showed a high resistance. The combination of these two products revealed then only an additive effect.

Sporulation Temperature Reveals a Requirement for CotE in the Assembly of both the Coat and Exosporium Layers of Bacillus cereus Spores.

PubMed

Bressuire-Isoard, Christelle; Bornard, Isabelle; Henriques, Adriano O; Carlin, Frédéric; Broussolle, Véronique

2016-01-01

The Bacillus cereus spore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers of B. cereus ATCC 14579 spores and on the resulting spore properties. The cotE deletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ΔcotE spores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ΔcotE spores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ΔcotE spores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H2O2 and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly of B. cereus spore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Intersporal Genetic Variation of Gigaspora margarita, a Vesicular Arbuscular Mycorrhizal Fungus, Revealed by M13 Minisatellite-Primed PCR.

PubMed

Zeze, A; Sulistyowati, E; Ophel-Keller, K; Barker, S; Smith, S

1997-02-01

Spores of vesicular arbuscular mycorrhizal (VAM) fungi contain thousands of nuclei. In order to understand the karyotic structure of a VAM fungus spore, the genetic variation of the first generation of spores from a VAM fungus (Gigaspora margarita) was examined. Spores originating from both single- and multispore inoculations of the species G. margarita were analyzed by M13 minisatellite-primed PCR. In both cases, different fingerprints were obtained from individual spores with few spores exhibiting similar fingerprints. These results can be explained only by a heterokaryotic status of the nuclear population within a spore.

[Survival of Bacillus anthracis spores in various tannery baths].

PubMed

Mendrycka, M; Mierzejewski, J

2000-01-01

The influence of tannery baths: liming, deliming, bating, pickling, tanning, retannage on the survival and on the germination dynamism of B. anthracis spores (Sterne strain) was investigated. The periods and the conditions of this influence were established according to technological process of cow hide tannage. Practically after every bath some part of the spores remained vital. The most effective killing of spores occurred after pickling, liming and deliming. Inversely, the most viable spores remained after bating and retannage process. The lack of correlation that was observed between survival and germination of spores after retannage bath can be explained by different mechanism of spores germination inhibition and their killing.

Global transcriptome analysis of spore formation in Myxococcus xanthus reveals a locus necessary for cell differentiation

PubMed Central

2010-01-01

Background Myxococcus xanthus is a Gram negative bacterium that can differentiate into metabolically quiescent, environmentally resistant spores. Little is known about the mechanisms involved in differentiation in part because sporulation is normally initiated at the culmination of a complex starvation-induced developmental program and only inside multicellular fruiting bodies. To obtain a broad overview of the sporulation process and to identify novel genes necessary for differentiation, we instead performed global transcriptome analysis of an artificial chemically-induced sporulation process in which addition of glycerol to vegetatively growing liquid cultures of M. xanthus leads to rapid and synchronized differentiation of nearly all cells into myxospore-like entities. Results Our analyses identified 1 486 genes whose expression was significantly regulated at least two-fold within four hours of chemical-induced differentiation. Most of the previously identified sporulation marker genes were significantly upregulated. In contrast, most genes that are required to build starvation-induced multicellular fruiting bodies, but which are not required for sporulation per se, were not significantly regulated in our analysis. Analysis of functional gene categories significantly over-represented in the regulated genes, suggested large rearrangements in core metabolic pathways, and in genes involved in protein synthesis and fate. We used the microarray data to identify a novel operon of eight genes that, when mutated, rendered cells unable to produce viable chemical- or starvation-induced spores. Importantly, these mutants displayed no defects in building fruiting bodies, suggesting these genes are necessary for the core sporulation process. Furthermore, during the starvation-induced developmental program, these genes were expressed in fruiting bodies but not in peripheral rods, a subpopulation of developing cells which do not sporulate. Conclusions These results suggest that microarray analysis of chemical-induced spore formation is an excellent system to specifically identify genes necessary for the core sporulation process of a Gram negative model organism for differentiation. PMID:20420673

Bicarbonate and amino acids are co-germinants for spores of Clostridium perfringens type A isolates carrying plasmid-borne enterotoxin gene.

PubMed

Alnoman, Maryam; Udompijitkul, Pathima; Banawas, Saeed; Sarker, Mahfuzur R

2018-02-01

Clostridium perfringens type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe) are generally linked to food poisoning, while isolates carrying cpe on a plasmid (P-cpe) are associated with non-food-borne gastrointestinal diseases. Both C-cpe and P-cpe isolates can form metabolically dormant spores, which through germination process return to actively growing cells to cause diseases. In our previous study, we showed that only 3 out of 20 amino acids (aa) in phosphate buffer (pH 7.0) triggered germination of spores of P-cpe isolates (P-cpe spores). We now found that 14 out of 20 individual aa tested induced germination of P-cpe spores in the presence of bicarbonate buffer (pH 7.0). However, no significant spore germination was observed with bicarbonate (pH 7.0) alone, indicating that aa and bicarbonate are co-germinants for P-cpe spores. P-cpe strain F4969 gerKC spores did not germinate, and gerAA spores germinated extremely poorly as compared to wild-type and gerKA spores with aa-bicarbonate (pH 7.0) co-germinants. The germination defects in gerKC and gerAA spores were partially restored by complementing gerKC or gerAA spores with wild-type gerKC or gerAA, respectively. Collectively, this study identified aa-bicarbonate as a novel nutrient germinant for P-cpe spores and provided evidence that GerKC and GerAA play major roles in aa-bicarbonate induced germination. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of DNA protection and repair in resistance of Bacillus subtilis spores to ultrahigh shock pressures simulating hypervelocity impacts.

PubMed

Moeller, Ralf; Horneck, Gerda; Rabbow, Elke; Reitz, Günther; Meyer, Cornelia; Hornemann, Ulrich; Stöffler, Dieter

2008-11-01

Impact-induced ejections of rocks from planetary surfaces are frequent events in the early history of the terrestrial planets and have been considered as a possible first step in the potential interplanetary transfer of microorganisms. Spores of Bacillus subtilis were used as a model system to study the effects of a simulated impact-caused ejection on rock-colonizing microorganisms using a high-explosive plane wave setup. Embedded in different types of rock material, spores were subjected to extremely high shock pressures (5 to 50 GPa) lasting for fractions of microseconds to seconds. Nearly exponential pressure response curves were obtained for spore survival and linear dependency for the induction of sporulation-defective mutants. Spores of strains defective in major small, acid-soluble spore proteins (SASP) (alpha/beta-type SASP) that largely protect the spore DNA and spores of strains deficient in nonhomologous-end-joining DNA repair were significantly more sensitive to the applied shock pressure than were wild-type spores. These results indicate that DNA may be the sensitive target of spores exposed to ultrahigh shock pressures. To assess the nature of the critical physical parameter responsible for spore inactivation by ultrahigh shock pressures, the resulting peak temperature was varied by lowering the preshock temperature, changing the rock composition and porosity, or increasing the water content of the samples. Increased peak temperatures led to increased spore inactivation and reduced mutation rates. The data suggested that besides the potential mechanical stress exerted by the shock pressure, the accompanying high peak temperatures were a critical stress parameter that spores had to cope with.

β-1,6-glucan synthesis-associated genes are required for proper spore wall formation in Saccharomyces cerevisiae.

PubMed

Pan, Hua-Ping; Wang, Ning; Tachikawa, Hiroyuki; Nakanishi, Hideki; Gao, Xiao-Dong

2017-11-01

The yeast spore wall is an excellent model to study the assembly of an extracellular macromolecule structure. In the present study, mutants defective in β-1,6-glucan synthesis, including kre1∆, kre6∆, kre9∆ and big1∆, were sporulated to analyse the effect of β-1,6-glucan defects on the spore wall. Except for kre6∆, these mutant spores were sensitive to treatment with ether, suggesting that the mutations perturb the integrity of the spore wall. Morphologically, the mutant spores were indistinguishable from wild-type spores. They lacked significant sporulation defects partly because the chitosan layer, which covers the glucan layer, compensated for the damage. The proof for this model was obtained from the effect of the additional deletion of CHS3 that resulted in the absence of the chitosan layer. Among the double mutants, the most severe spore wall deficiency was observed in big1∆ spores. The majority of the big1∆chs3∆ mutants failed to form visible spores at a higher temperature. Given that the big1∆ mutation caused a failure to attach a GPI-anchored reporter, Cwp2-GFP, to the spore wall, β-1,6-glucan is involved in tethering of GPI-anchored proteins in the spore wall as well as in the vegetative cell wall. Thus, β-1,6-glucan is required for proper organization of the spore wall. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Apparatus and method for automated monitoring of airborne bacterial spores

NASA Technical Reports Server (NTRS)

Ponce, Adrian (Inventor)

2009-01-01

An apparatus and method for automated monitoring of airborne bacterial spores. The apparatus is provided with an air sampler, a surface for capturing airborne spores, a thermal lysis unit to release DPA from bacterial spores, a source of lanthanide ions, and a spectrometer for excitation and detection of the characteristic fluorescence of the aromatic molecules in bacterial spores complexed with lanthanide ions. In accordance with the method: computer-programmed steps allow for automation of the apparatus for the monitoring of airborne bacterial spores.

Requirements for the Development of Bacillus Anthracis Spore Reference Materials Used to Test Detection Systems

DTIC Science & Technology

2006-01-01

the sporangium) contributes the com- plex layers of the spore coats that encase the spore DNA. The mother cell dies and begins to fall apart at the end...spores. Bacillus spores contain a number of coat layers and some species posses an additional outermost layer called the exosporium. BA, B. cereus, and B...exosporium is the outermost layer of the BA spores, it likely contains important protein and carbohydrate markers that are recognized by antibodies

Effect of Fertilizers and Neem Cake Amendment in Soil on Spore Germination of Arthrobotrys dactyloides

PubMed Central

Kumar, D.; Jaiswal, R. K.

2005-01-01

Application of fertilizers such as urea, diammonium phosphate (DAP) and muriate of potash in soil adversely affected the spore germination of Arthrobotrys dactyloides. Amendment of soil with urea at the concentrations of 1.0%, 0.5% and 0.1% completely inhibited spore germination and direct trap formation on the conidium, whereas muriate of potash delayed and reduced the spore germination even at the lowest concentration. DAP also inhibited spore germination at 1.0% concentration, while at lower concentration the percentage of spore germination was reduced. Application of neem cake at the concentration of 0.5% also inhibited spore germination after 24 h of amendment. The inhibitory effect of neem cake was reduced after 15 days of amendment, while after 30 days after amendment the inhibitory effect was completely lost and the spore germinated by direct trap as in unamended soil. Nematodes were not attracted to ungerminated spores after 24 h of amendment. After 15 days of amendment nematodes were attracted to agar blocks containing fewer germinated spores after 24 h of incubation but after 48 h of incubation large number of nematodes were attracted and trapped by the germinated spores with direct traps. After 30 days of amendment, larger number of nematodes were attracted and trapped by direct traps. PMID:24049500

Spore-to-spore agar culture of the myxomycete Physarum globuliferum.

PubMed

Liu, Pu; Wang, Qi; Li, Yu

2010-02-01

The ontogeny of the myxomycete Physarum globuliferum was observed on corn meal agar and hanging drop cultures without adding sterile oat flakes, bacteria or other microorganisms. Its complete life cycle including spore germination, myxamoebae, swarm cells, plasmodial development, and maturity of fructifications was demonstrated. Details of spore-to-spore development are described and illustrated.

Bacterial spore inactivation induced by cold plasma.

PubMed

Liao, Xinyu; Muhammad, Aliyu Idris; Chen, Shiguo; Hu, Yaqin; Ye, Xingqian; Liu, Donghong; Ding, Tian

2018-04-05

Cold plasma has emerged as a non-thermal technology for microbial inactivation in the food industry over the last decade. Spore-forming microorganisms pose challenges for microbiological safety and for the prevention of food spoilage. Inactivation of spores induced by cold plasma has been reported by several studies. However, the exact mechanism of spore deactivation by cold plasma is poorly understood; therefore, it is difficult to control this process and to optimize cold plasma processing for efficient spore inactivation. In this review, we summarize the factors that affect the resistance of spores to cold plasma, including processing parameters, environmental elements, and spore properties. We then describe possible inactivation targets in spore cells (e.g., outer structure, DNA, and metabolic proteins) that associated with inactivation by cold plasma according to previous studies. Kinetic models of the sporicidal activity of cold plasma have also been described here. A better understanding of the interaction between spores and cold plasma is essential for the development and optimization of cold plasma technology in food the industry.

A side-by-side comparison of Rotorod and Burkard pollen and spore collections.

PubMed

Crisp, Howard C; Gomez, Robert A; White, Kevin M; Quinn, James M

2013-08-01

The Rotorod sampler and Burkard spore trap are 2 devices commonly used to quantify airborne particles. To evaluate the differences in collections between the 2 devices for a wide range of plant pollens and fungal spores. Pollens and spores were collected simultaneously with each device on 167 days during a 1-year period. The Burkard yielded significantly higher total and individual mold spore counts. It yielded statistically higher total grass, total weed, and Urticaceae daily pollen counts, although the absolute differences were small. Daily counts were positively correlated between the 2 devices for the most abundant pollens and mold spores. The Burkard spore trap collects many more mold spores than the Rotorod over a wide variety of species. The Burkard also yielded higher total grass, total weed, and Urticaceae daily pollen counts. Despite these differences, however, either device can be used to follow trends in the most abundant pollen and mold spores. Copyright © 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Biological indicators for steam sterilization: characterization of a rapid biological indicator utilizing Bacillus stearothermophilus spore-associated alpha-glucosidase enzyme.

PubMed

Albert, H; Davies, D J; Woodson, L P; Soper, C J

1998-11-01

The alpha-glucosidase enzyme was isolated from vegetative cells and spores of Bacillus stearothermophilus, ATCC 7953. Spore-associated enzyme had a molecular weight of approximately 92,700, a temperature optimum of 60 degrees C, and a pH optimum of 7.0-7.5. The enzyme in crude aqueous spore extract was stable for 30 min up to a temperature of 65 degrees C, above which the enzyme was rapidly denatured. The optimal pH for stability of the enzyme was approximately 7.2. The alpha-glucosidase in crude vegetative cell extract had similar characteristics to the spore-associated enzyme but its molecular weight was 86,700. The vegetative cell and spore-associated enzymes were cross-reactive. The enzymes are postulated to derive from a single gene product, which undergoes modification to produce the spore-associated form. The location of alpha-glucosidase in the spore coats (outside the spore protoplast) is consistent with the location of most enzymes involved in activation, germination and outgrowth.

The effect of meteorological factors on the daily variation of airborne fungal spores in Granada (southern Spain)

NASA Astrophysics Data System (ADS)

Sabariego, S.; Díaz de la Guardia, C.; Alba, F.

A study was made of the link between climatic factors and the daily content of certain fungal spores in the atmosphere of the city of Granada in 1994. Sampling was carried out with a Burkard 7-day-recording spore trap. The spores analysed corresponded to the taxa Alternaria, Ustilago and Cladosporium, with two morphologically different spore types in the latter genus, cladosporioides and herbarum. These spores were selected both for their allergenic capacity and for the high level of their presence in the atmosphere, particularly during the spring and autumn. The spores of Cladosporium were the most abundant (93.82% of the total spores identified). The Spearman correlation coefficients between the spore concentrations studied and the meteorological parameters show different indices depending on the taxon being analysed. Alternaria and Cladosporium are significantly correlated with temperature and hours of sunlight, while Ustilago shows positive correlation indices with relative humidity and negative indices with wind speed.

Detect-to-treat: development of analysis of bacilli spores in nasal mucus by surfaced-enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

Inscore, Frank E.; Gift, Alan D.; Farquharson, Stuart

2004-12-01

As the war on terrorism in Afghanistan and Iraq continue, future attacks both abroad and in the U.S.A. are expected. In an effort to aid civilian and military personnel, we have been investigating the potential of using a surface-enhanced Raman spectroscopy (SERS) sampling device to detect Bacillus anthracis spores in nasal swab samples. Such a device would be extremely beneficial to medical responders and management in assessing the extent of a bioterrorist attack and making detect-to-treat decisions. The disposable sample device consists of a glass capillary filled with a silver-doped sol-gel that is capable of extracting dipicolinic acid (DPA), a chemical signature of Bacilli, and generating SERS spectra. The sampling device and preliminary measurements of DPA extracted from spores and nasal mucus will be presented.

Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-specific genes

PubMed Central

Galperin, Michael Y; Mekhedov, Sergei L; Puigbo, Pere; Smirnov, Sergey; Wolf, Yuri I; Rigden, Daniel J

2012-01-01

Three classes of low-G+C Gram-positive bacteria (Firmicutes), Bacilli, Clostridia and Negativicutes, include numerous members that are capable of producing heat-resistant endospores. Spore-forming firmicutes include many environmentally important organisms, such as insect pathogens and cellulose-degrading industrial strains, as well as human pathogens responsible for such diseases as anthrax, botulism, gas gangrene and tetanus. In the best-studied model organism Bacillus subtilis, sporulation involves over 500 genes, many of which are conserved among other bacilli and clostridia. This work aimed to define the genomic requirements for sporulation through an analysis of the presence of sporulation genes in various firmicutes, including those with smaller genomes than B. subtilis. Cultivable spore-formers were found to have genomes larger than 2300 kb and encompass over 2150 protein-coding genes of which 60 are orthologues of genes that are apparently essential for sporulation in B. subtilis. Clostridial spore-formers lack, among others, spoIIB, sda, spoVID and safA genes and have non-orthologous displacements of spoIIQ and spoIVFA, suggesting substantial differences between bacilli and clostridia in the engulfment and spore coat formation steps. Many B. subtilis sporulation genes, particularly those encoding small acid-soluble spore proteins and spore coat proteins, were found only in the family Bacillaceae, or even in a subset of Bacillus spp. Phylogenetic profiles of sporulation genes, compiled in this work, confirm the presence of a common sporulation gene core, but also illuminate the diversity of the sporulation processes within various lineages. These profiles should help further experimental studies of uncharacterized widespread sporulation genes, which would ultimately allow delineation of the minimal set(s) of sporulation-specific genes in Bacilli and Clostridia. PMID:22882546

The effects of probiotic, prebiotic and synbiotic diets containing Bacillus coagulans and inulin on rat intestinal microbiota

PubMed Central

Abhari, Kh; Shekarforoush, S. S; Sajedianfard, J; Hosseinzadeh, S; Nazifi, S

2015-01-01

An in vivo experiment was conducted to study the effects of probiotic Bacillus coagulans spores, with and without prebiotic, inulin, on gastrointestinal (GI) microbiota of healthy rats and its potentiality to survive in the GI tract. Forty-eight male Wistar rats were randomly divided into four groups (n=12) and fed as follows: standard diet (control), standard diet supplied with 5% w/w long chain inulin (prebiotic), standard diet with 109/day spores of B. coagulans by orogastric gavage (probiotic), and standard diet with 5% w/w long chain inulin and 109 spores/day of B. coagulans by orogastric gavage (synbiotic). Rats were fed the diets for 30 days. At day 10, 20 and 30 of experiment, 24 h post administration, four rats from each group were randomly selected and after faecal collection were sacrificed. Small intestine, cecum, and colon were excised from each rat and used for microbial analysis. Administration of synbiotic and probiotic diets led to a significant (P<0.05) increment in lactic acid bacteria (LAB), total aerobic and total anaerobic population compared the prebiotic and control diets. A significant decrease in Enterobacteriaceae counts of various segments of GI tract (except small intestine) in synbiotic, probiotic and prebiotic fed groups were also seen. The obvious decline in spores count through passing GI tract and high surviving spore counts in faecal samples showed that spores are not a normal resident of GI microbiota and affect intestinal microbiota by temporary proliferation. In conclusion, the present study clearly showed probiotic B. coagulans was efficient in beneficially modulating GI microbiota and considering transitional characteristics of B. coagulans, daily consumption of probiotic products is necessary for any long-term effect. PMID:27175187

Grapefruit extract activity in the control of rose powdery mildew and black spot.

PubMed

Wojdyła, A T

2001-01-01

Efficacy of grapefruit extract (a.i. of Biosept 33 SL) in the control of Sphaerotheca pannosa var. rosae and Diplocarpon rosae on roses was investigated during 1998-1999. The extract was applied as plant spray in concentrations from 0.017 to 0.099%. First treatment of rose shrubs was done when visible disease symptoms occurred on leaves and spraying was repeated 3 (in plastic tunnel) or 10-times (in the field) at weekly intervals. In the second experiment roses with visible powdery mildew symptoms were sprayed once with grapefruit extract. Leaves were sampled one or 7 days after the extract application and germination of spores of S. pannosa var. rosae on potato dextrose agar was evaluated. In the next experiment roses grown under plastic tunnel were sprayed once with the tested preparation. After 24 hours leaves were collected and appearance of fungal hyphae and spores of S. pannosa var. rosae was studied in scanning electron microscope. In the control of S. pannosa var. rosae grapefruit extract at conc. 0.066% was as effective as triforine (standard) applied at 0.027%. Reduction of concentration resulted in the decreased efficacy of the tested preparation. Spores of S. pannosa var. rosae collected one day after grapefruit extract application germinated in about 5%. Analyses of spore vitality 6 days letter showed that only about 15% of conidia could germinated on PDA agar. In contrary, spores from untreated leaves germinated in about 95%. Scanning electrone microscope analysis of leaves taken from plants protected with grapefruit extract showed that most of hyphae were separated from leaf surface. Almost all hyphae and spores were degenerated. In the control of D. rosae the preparation in all tested concentrations gave satisfactory results but was less effective than triforine.

The effects of probiotic, prebiotic and synbiotic diets containing Bacillus coagulans and inulin on rat intestinal microbiota.

PubMed

Abhari, Kh; Shekarforoush, S S; Sajedianfard, J; Hosseinzadeh, S; Nazifi, S

2015-01-01

An in vivo experiment was conducted to study the effects of probiotic Bacillus coagulans spores, with and without prebiotic, inulin, on gastrointestinal (GI) microbiota of healthy rats and its potentiality to survive in the GI tract. Forty-eight male Wistar rats were randomly divided into four groups (n=12) and fed as follows: standard diet (control), standard diet supplied with 5% w/w long chain inulin (prebiotic), standard diet with 10(9)/day spores of B. coagulans by orogastric gavage (probiotic), and standard diet with 5% w/w long chain inulin and 10(9) spores/day of B. coagulans by orogastric gavage (synbiotic). Rats were fed the diets for 30 days. At day 10, 20 and 30 of experiment, 24 h post administration, four rats from each group were randomly selected and after faecal collection were sacrificed. Small intestine, cecum, and colon were excised from each rat and used for microbial analysis. Administration of synbiotic and probiotic diets led to a significant (P<0.05) increment in lactic acid bacteria (LAB), total aerobic and total anaerobic population compared the prebiotic and control diets. A significant decrease in Enterobacteriaceae counts of various segments of GI tract (except small intestine) in synbiotic, probiotic and prebiotic fed groups were also seen. The obvious decline in spores count through passing GI tract and high surviving spore counts in faecal samples showed that spores are not a normal resident of GI microbiota and affect intestinal microbiota by temporary proliferation. In conclusion, the present study clearly showed probiotic B. coagulans was efficient in beneficially modulating GI microbiota and considering transitional characteristics of B. coagulans, daily consumption of probiotic products is necessary for any long-term effect.

Optical and structural properties of plasma-treated Cordyceps bassiana spores as studied by circular dichroism, absorption, and fluorescence spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Geon Joon, E-mail: gjlee@kw.ac.kr; Sim, Geon Bo; Choi, Eun Ha

To understand the killing mechanism of fungal spores by plasma treatment, the optical, structural, and biological properties of the insect pathogenic fungus Cordyceps bassiana spores were studied. A nonthermal atmospheric-pressure plasma jet (APPJ) was used to treat the spores in aqueous solution. Optical emission spectra of the APPJ acquired in air indicated emission peaks corresponding to hydroxyl radicals and atomic oxygen. When the APPJ entered the aqueous solution, additional reactive species were derived from the interaction of plasma radicals with the aqueous solution. Fluorescence and absorption spectroscopy confirmed the generation of hydroxyl radicals and hydrogen peroxide in the plasma-activated watermore » (PAW). Spore counting showed that plasma treatment significantly reduced spore viability. Absorption spectroscopy, circular dichroism (CD) spectroscopy, and agarose gel electrophoresis of the DNA extracted from plasma-treated spores showed a reduction in spore DNA content. The magnitude of the dip in the CD spectrum was lower in the plasma-treated spores than in the control, indicating that plasma treatment causes structural modifications and/or damage to cellular components. Tryptophan fluorescence intensity was lower in the plasma-treated spores than in the control, suggesting that plasma treatment modified cell wall proteins. Changes in spore viability and DNA content were attributed to structural modification of the cell wall by reactive species coming from the APPJ and the PAW. Our results provided evidence that the plasma radicals and the derived reactive species play critical roles in fungal spore inactivation.« less

Optical and structural properties of plasma-treated Cordyceps bassiana spores as studied by circular dichroism, absorption, and fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Lee, Geon Joon; Sim, Geon Bo; Choi, Eun Ha; Kwon, Young-Wan; Kim, Jun Young; Jang, Siun; Kim, Seong Hwan

2015-01-01

To understand the killing mechanism of fungal spores by plasma treatment, the optical, structural, and biological properties of the insect pathogenic fungus Cordyceps bassiana spores were studied. A nonthermal atmospheric-pressure plasma jet (APPJ) was used to treat the spores in aqueous solution. Optical emission spectra of the APPJ acquired in air indicated emission peaks corresponding to hydroxyl radicals and atomic oxygen. When the APPJ entered the aqueous solution, additional reactive species were derived from the interaction of plasma radicals with the aqueous solution. Fluorescence and absorption spectroscopy confirmed the generation of hydroxyl radicals and hydrogen peroxide in the plasma-activated water (PAW). Spore counting showed that plasma treatment significantly reduced spore viability. Absorption spectroscopy, circular dichroism (CD) spectroscopy, and agarose gel electrophoresis of the DNA extracted from plasma-treated spores showed a reduction in spore DNA content. The magnitude of the dip in the CD spectrum was lower in the plasma-treated spores than in the control, indicating that plasma treatment causes structural modifications and/or damage to cellular components. Tryptophan fluorescence intensity was lower in the plasma-treated spores than in the control, suggesting that plasma treatment modified cell wall proteins. Changes in spore viability and DNA content were attributed to structural modification of the cell wall by reactive species coming from the APPJ and the PAW. Our results provided evidence that the plasma radicals and the derived reactive species play critical roles in fungal spore inactivation.

The effects of meteorological factors on airborne fungal spore concentration in two areas differing in urbanisation level

NASA Astrophysics Data System (ADS)

Oliveira, M.; Ribeiro, H.; Delgado, J. L.; Abreu, I.

2009-01-01

Although fungal spores are an ever-present component of the atmosphere throughout the year, their concentration oscillates widely. This work aims to establish correlations between fungal spore concentrations in Porto and Amares and meteorological data. The seasonal distribution of fungal spores was studied continuously (2005-2007) using volumetric spore traps. To determine the effect of meteorological factors (temperature, relative humidity and rainfall) on spore concentration, the Spearman rank correlation test was used. In both locations, the most abundant fungal spores were Cladosporium, Agaricus, Agrocybe, Alternaria and Aspergillus/Penicillium, the highest concentrations being found during summer and autumn. In the present study, with the exception of Coprinus and Pleospora, spore concentrations were higher in the rural area than in the urban location. Among the selected spore types, spring-autumn spores ( Coprinus, Didymella, Leptosphaeria and Pleospora) exhibited negative correlations with temperature and positive correlations both with relative humidity and rainfall level. On the contrary, late spring-early summer (Smuts) and summer spores ( Alternaria, Cladosporium, Epicoccum, Ganoderma, Stemphylium and Ustilago) exhibited positive correlations with temperature and negative correlations both with relative humidity and rainfall level. Rust, a frequent spore type during summer, had a positive correlation with temperature. Aspergillus/Penicillium, showed no correlation with the meteorological factors analysed. This knowledge can be useful for agriculture, allowing more efficient and reliable application of pesticides, and for human health, by improving the diagnosis and treatment of respiratory allergic disease.

Photometric immersion refractometry of bacterial spores.

PubMed Central

Gerhardt, P; Beaman, T C; Corner, T R; Greenamyre, J T; Tisa, L S

1982-01-01

Photometric immersion refractometry was used to determine the average apparent refractive index (n) of five types of dormant Bacillus spores representing a 600-fold range in moist-heat resistance determined as a D100 value. The n of a spore type increased as the molecular size of various immersion solutes decreased. For comparison of the spore types, the n of the entire spore and of the isolated integument was determined by use of bovine serum albumin, which is excluded from permeating into them. The n of the sporoplast (the structures bounded by the outer pericortex membrane) was determined by use of glucose, which was shown to permeate into the spore only as deeply as the pericortex membrane. Among the various spore types, an exponential increase in the heat resistance correlated with the n of the entire spore and of the sporoplast, but not of the isolated perisporoplast integument. Correlation of the n with the solids content of the entire spore provided a method of experimentally obtaining the refractive index increment (dn/dc), which was constant for the various spore types and enables the calculation of solids and water content from an n. Altogether, the results showed that the total water content is distributed unequally within the dormant spore, with less water in the sporoplast than in the perisporoplast integument, and that the sporoplast becomes more refractile and therefore more dehydrated as the heat resistance becomes greater among the various spore types. PMID:6802796

Architecture and High-Resolution Structure of Bacillus thuringiensis and Bacillus cereus Spore Coat Surfaces

DOE Office of Scientific and Technical Information (OSTI.GOV)

Plomp, M; Leighton, T; Wheeler, K

2005-02-18

We have utilized atomic force microscopy (AFM) to visualize the native surface topology and ultrastructure of Bacillus thuringiensis and Bacillus cereus spores in water and in air. AFM was able to resolve the nanostructure of the exosporium and three distinctive classes of appendages. Removal of the exosporium exposed either a hexagonal honeycomb layer (B. thuringiensis) or a rodlet outer spore coat layer (B. cereus). Removal of the rodlet structure from B. cereus spores revealed an underlying honeycomb layer similar to that observed with B. thuringiensis spores. The periodicity of the rodlet structure on the outer spore coat of B. cereusmore » was {approx}8 nm, and the length of the rodlets was limited to the cross-patched domain structure of this layer to {approx}200 nm. The lattice constant of the honeycomb structures was {approx}9 nm for both B. cereus and B. thuringiensis spores. Both honeycomb structures were composed of multiple, disoriented domains with distinct boundaries. Our results demonstrate that variations in storage and preparation procedures result in architectural changes in individual spore surfaces, which establish AFM as a useful tool for evaluation of preparation and processing ''fingerprints'' of bacterial spores. These results establish that high-resolution AFM has the capacity to reveal species-specific assembly and nanometer scale structure of spore surfaces. These species-specific spore surface structural variations are correlated with sequence divergences in a spore core structural protein SspE.« less

Dipicolinic Acid Release by Germinating Clostridium difficile Spores Occurs through a Mechanosensing Mechanism.

PubMed

Francis, Michael B; Sorg, Joseph A

2016-01-01

Classically, dormant endospores are defined by their resistance properties, particularly their resistance to heat. Much of the heat resistance is due to the large amount of dipicolinic acid (DPA) stored within the spore core. During spore germination, DPA is released and allows for rehydration of the otherwise-dehydrated core. In Bacillus subtilis , 7 proteins are encoded by the spoVA operon and are important for DPA release. These proteins receive a signal from the activated germinant receptor and release DPA. This DPA activates the cortex lytic enzyme CwlJ, and cortex degradation begins. In Clostridium difficile , spore germination is initiated in response to certain bile acids and amino acids. These bile acids interact with the CspC germinant receptor, which then transfers the signal to the CspB protease. Activated CspB cleaves the cortex lytic enzyme, pro-SleC, to its active form. Subsequently, DPA is released from the core. C. difficile encodes orthologues of spoVAC , spoVAD , and spoVAE . Of these, the B. subtilis SpoVAC protein was shown to be capable of mechanosensing. Because cortex degradation precedes DPA release during C. difficile spore germination (opposite of what occurs in B. subtilis ), we hypothesized that cortex degradation would relieve the osmotic constraints placed on the inner spore membrane and permit DPA release. Here, we assayed germination in the presence of osmolytes, and we found that they can delay DPA release from germinating C. difficile spores while still permitting cortex degradation. Together, our results suggest that DPA release during C. difficile spore germination occurs though a mechanosensing mechanism. IMPORTANCE Clostridium difficile is transmitted between hosts in the form of a dormant spore, and germination by C. difficile spores is required to initiate infection, because the toxins that are necessary for disease are not deposited on the spore form. Importantly, the C. difficile spore germination pathway represents a novel pathway for bacterial spore germination. Prior work has shown that the order of events during C. difficile spore germination (cortex degradation and DPA release) is flipped compared to the events during B. subtilis spore germination, a model organism. Here, we further characterize the C. difficile spore germination pathway and summarize our findings indicating that DPA release by germinating C. difficile spores occurs through a mechanosensing mechanism in response to the degradation of the spore cortex.

Using thermal inactivation kinetics to calculate the probability of extreme spore longevity: implications for paleomicrobiology and lithopanspermia.

PubMed

Nicholson, Wayne L

2003-12-01

Thermal inactivation kinetics with extrapolation were used to model the survival probabilities of spores of various Bacillus species over time periods of millions of years at the historical ambient temperatures (25-40 degrees C) encountered within the 250 million-year-old Salado formation, from which the putative ancient spore-forming bacterium Salibacillus marismortui strain 2-9-3 was recovered. The model indicated extremely low-to-moderate survival probabilities for spores of mesophiles. but surprisingly high survival probabilities for thermophilic spores. The significance of the results are discussed in terms of the survival probabilities of (i) terrestrial spores in ancient geologic samples and (ii) spores transported between planets within impact ejecta.

Using Thermal Inactivation Kinetics to Calculate the Probability of Extreme Spore Longevity: Implications for Paleomicrobiology and Lithopanspermia

NASA Astrophysics Data System (ADS)

Nicholson, Wayne L.

2003-12-01

Thermal inactivation kinetics with extrapolation were used to model the survival probabilities of spores of various Bacillus species over time periods of millions of years at the historical ambient temperatures (25-40 °) encountered within the 250 million-year-old Salado formation, from which the putative ancient spore-forming bacterium Salibacillus marismortui strain 2-9-3 was recovered. The model indicated extremely low-to-moderate survival probabilities for spores of mesophiles, but surprisingly high survival probabilities for thermophilic spores. The significance of the results are discussed in terms of the survival probabilities of (i) terrestrial spores in ancient geologic samples and (ii) spores transported between planets within impact ejecta.

Ethyl p-coumarate exerts antifungal activity in vitro and in vivo against fruit Alternaria alternata via membrane-targeted mechanism.

PubMed

Li, Wusun; Yuan, Shuzhi; Sun, Jing; Li, Qianqian; Jiang, Weibo; Cao, Jiankang

2018-08-02

The fungus Alternaria alternata can cause food contamination by black spot rot and food safety issues due to the production of mycotoxins. In particular, A. alternata can infect many fresh fruits and vegetables and lead to considerable postharvest decay during storage and processing. The use of plant-derived products in postharvest disease management may be an acceptable alternative to traditional chemical fungicides. The aim of this study was to assess the antifungal activity of ethyl p-coumarate (EpCA) against Alternaria alternata in vitro and in vivo, and to determine the underlying mechanism. Results indicated that EpCA exhibited pronounced antifungal activity against in vitro mycelial growth of A. alternata, with half-inhibition concentration of 176.8 μg/mL. Spore germination of the pathogen was inhibited by EpCA in a dose-dependent manner. Moreover, in vivo test confirmed that both 100 and 800 μg/mL EpCA significantly reduced disease development of black spot rot in jujube fruit caused by A. alternata. The EpCA treatments increased plasma membrane permeability as great leakage of intercellular electrolytes, soluble proteins and sugars of A. alternata occurred during incubation. The EpCA treatments also caused increase of the influx of propidium iodide, a fluorescence dye binding nucleus DNA, into the affected spores, indicating the disrupted plasma membrane integrity. Observations of ultrastructure further evidenced the damage to plasma membrane and morphology of A. alternata caused by EpCA, which resulted in distortion, sunken and shrivelled of spores and mycelia of the pathogen. In addition, fluorometric assay by confocal laser scanning microscopy confirmed that the EpCA treatments induced endogenous reactive oxygen species (ROS) formation in the spores of A. alternata, with stronger and more stable accumulation of ROS at higher concentration of EpCA. Therefore, heavy oxidative damage to cellular membranes and organelles might happen as demonstrated by the severe occurrence of lipid peroxidation of the pathogen treated with EpCA. Taken together, these results indicated that EpCA exerts antifungal activity via membrane-targeted mechanism and it would be a promising candidate to control postharvest diseases of fruits. Copyright © 2018 Elsevier B.V. All rights reserved.

Measurement of Metabolic Activity in Dormant Spores of Bacillus Species

DTIC Science & Technology

2015-01-14

SECURITY CLASSIFICATION OF: Spores of Bacillus megaterium and Bacillus subtilis were harvested shortly after release from sporangia, incubated under...Measurement of Metabolic Activity in Dormant Spores of Bacillus Species Report Title Spores of Bacillus megaterium and Bacillus subtilis were...ribosomal RNA when newly harvested Bacillus subtilis spores are incubated at physiological temperatures, as well as some evidence for transcription in

Ecology and thermal inactivation of microbes in and on interplanetary space vehicle components

NASA Technical Reports Server (NTRS)

Campbell, J. E.; Reyes, A. L.; Wehby, A. J.; Crawford, R. G.; Wimsatt, J. C.; Peeler, J. T.

1974-01-01

Dry heat sterilization of spacecraft was investigated by studying the production of spore crops, and thermal inactivation of the spores, and bacillus subtillus. Spore assays were made by conventional plate count methods, and survival curves for the spores are presented. The results indicate that the inherent resistance of spores from a parent cell can be maintained.

Validation of Respirator Filter Efficacy

DTIC Science & Technology

2003-03-20

microorganism Bacillus atrophaeus formerly Bacillus globigii or BG). The BG spore of approximately 1 µm diameter and inert particles over a range...conducted using the spore form of the microorganism Bacillus atrophaeus (formerly Bacillus globigii or BG). The BG spore is elliptically shaped with...will be conducted using the spore form of the microorganism Bacillus atrophaeus (formerly Bacillus globigii or BG). The BG spore is elliptically

Decontamination assessment of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surfaces using a hydrogen peroxide gas generator.

PubMed

Rogers, J V; Sabourin, C L K; Choi, Y W; Richter, W R; Rudnicki, D C; Riggs, K B; Taylor, M L; Chang, J

2005-01-01

To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. Bacillus anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to > or =1000 ppm hydrogen peroxide gas for 20 min. Hydrogen peroxide exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials except G. stearothermophilus on industrial carpet. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with both surrogates. The effectiveness of gaseous hydrogen peroxide on the growth of biological indicators and spore strips was evaluated in parallel as a qualitative assessment of decontamination. At 1 and 7 days postexposure, decontaminated biological indicators and spore strips exhibited no growth, while the nondecontaminated samples displayed growth. Significant differences in decontamination efficacy of hydrogen peroxide gas on porous and nonporous surfaces were observed when comparing the mean log reduction in B. anthracis spores with B. subtilis and G. stearothermophilus spores. These results provide comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using hydrogen peroxide gas.

Kinetics of Germination of Individual Spores of Geobacillus stearothermophilus as Measured by Raman Spectroscopy and Differential Interference Contrast Microscopy

PubMed Central

Zhou, Tingting; Dong, Zhiyang; Setlow, Peter; Li, Yong-qing

2013-01-01

Geobacillus stearothermophilus is a gram-positive, thermophilic bacterium, spores of which are very heat resistant. Raman spectroscopy and differential interference contrast microscopy were used to monitor the kinetics of germination of individual spores of G. stearothermophilus at different temperatures, and major conclusions from this work were as follows. 1) The CaDPA level of individual G. stearothermophilus spores was similar to that of Bacillus spores. However, the Raman spectra of protein amide bands suggested there are differences in protein structure in spores of G. stearothermophilus and Bacillus species. 2) During nutrient germination of G. stearothermophilus spores, CaDPA was released beginning after a lag time (T lag) between addition of nutrient germinants and initiation of CaDPA release. CaDPA release was complete at T release, and ΔT release (T release – T lag) was 1–2 min. 3) Activation by heat or sodium nitrite was essential for efficient nutrient germination of G. stearothermophilus spores, primarily by decreasing T lag values. 4) Values of T lag and T release were heterogeneous among individual spores, but ΔT release values were relatively constant. 5) Temperature had major effects on nutrient germination of G. stearothermophilus spores, as at temperatures below 65°C, average T lag values increased significantly. 6) G. stearothermophilus spore germination with exogenous CaDPA or dodecylamine was fastest at 65°C, with longer Tlag values at lower temperatures. 7) Decoating of G. stearothermophilus spores slowed nutrient germination slightly and CaDPA germination significantly, but increased dodecylamine germination markedly. These results indicate that the dynamics and heterogeneity of the germination of individual G. stearothermophilus spores are generally similar to that of Bacillus species. PMID:24058645

Effects of High Pressure on Bacillus licheniformis Spore Germination and Inactivation

PubMed Central

Borch-Pedersen, Kristina; Mellegård, Hilde; Reineke, Kai; Boysen, Preben; Sevenich, Robert; Lindbäck, Toril

2017-01-01

ABSTRACT Bacillus and Clostridium species form spores, which pose a challenge to the food industry due to their ubiquitous nature and extreme resistance. Pressurization at <300 MPa triggers spore germination by activating germination receptors (GRs), while pressurization at >300 MPa likely triggers germination by opening dipicolinic acid (DPA) channels present in the inner membrane of the spores. In this work, we expose spores of Bacillus licheniformis, a species associated with food spoilage and occasionally with food poisoning, to high pressure (HP) for holding times of up to 2 h. By using mutant spores lacking one or several GRs, we dissect the roles of the GerA, Ynd, and GerK GRs in moderately HP (mHP; 150 MPa)-induced spore germination. We show that Ynd alone is sufficient for efficient mHP-induced spore germination. GerK also triggers germination with mHP, although at a reduced germination rate compared to that of Ynd. GerA stimulates mHP-induced germination but only in the presence of either the intact GerK or Ynd GR. These results suggests that the effectiveness of the individual GRs in mHP-induced germination differs from their effectiveness in nutrient-induced germination, where GerA plays an essential role. In contrast to Bacillus subtilis spores, treatment with very HP (vHP) of 550 MPa at 37°C did not promote effective germination of B. licheniformis spores. However, treatment with vHP in combination with elevated temperatures (60°C) gave a synergistic effect on spore germination and inactivation. Together, these results provide novel insights into how HP affects B. licheniformis spore germination and inactivation and the role of individual GRs in this process. IMPORTANCE Bacterial spores are inherently resistant to food-processing regimes, such as high-temperature short-time pasteurization, and may therefore compromise food durability and safety. The induction of spore germination facilitates subsequent inactivation by gentler processing conditions that maintain the sensory and nutritional qualities of the food. High-pressure (HP) processing is a nonthermal food-processing technology used to eliminate microbes from food. The application of this technology for spore eradication in the food industry requires a better understanding of how HP affects the spores of different bacterial species. The present study provides novel insights into how HP affects Bacillus licheniformis spores, a species associated with food spoilage and occasionally food poisoning. We describe the roles of different germination receptors in HP-induced germination and the effects of two different pressure levels on the germination and inactivation of spores. This study will potentially contribute to the effort to implement HP technology for spore inactivation in the food industry. PMID:28476768

Ecology and thermal inactivation of microbes in and on interplanetary space vehicle components

NASA Technical Reports Server (NTRS)

Campbell, J. E.

1973-01-01

Studies were made of atypical organisms found in Bacillus subtilis var. niger spore colonies. Efforts were aimed at: (1) determining the heat sensitivity of these atypical white spores treated under dry heat conditions and their influence on the nature of the survival curve, (2) preparing a new spore crop obtained from spore isolates by purification procedures, and (3) comparing spore crops obtained from Cape Kennedy (SSM-10) and Minnesota (Minn. sp. AAEF) with the old Cincinnati and new purified Cincinnati spore crop under dry heat conditions.

The trace analysis of microorganisms in real samples by combination of a filtration microcartridge and capillary isoelectric focusing.

PubMed

Horká, Marie; Horký, Jaroslav; Kubesová, Anna; Zapletalová, Eva; Slais, Karel

2011-07-01

Trace analysis of microorganisms in real biological samples needs very sensitive methods for their detection. Most procedures for detecting and quantifying pathogens require a sample preparation step including concentrating microorganisms from large sample volumes with high and reproducible efficiency. Electromigration techniques have great potential to include the preconcentration, separation, and detection of whole cells and therefore they can rapidly indicate the presence of pathogens. The preconcentration and separation of microorganisms from real suspensions utilising a combination of filtration and capillary isoelectric focusing was developed and the possibility for its application to real samples was verified. For our experiments, spores of Monilinia species and of Penicillium expansum were selected as model bioparticles, as they cause major losses in agrosystems. The isoelectric points of the spores of M. laxa, M. fructigena, M. fruticola, and P. expansum were determined and the method was verified using real samples taken directly from infected apples. The coupling of a filtration cartridge with a separation capillary can improve the detection limit of isoelectric focusing with UV detection by at least 4 orders of magnitude. Spores of M. fructigena and of M. laxa in numbers of hundreds of particles per milliliter were detected on a visually noninfected apple surface which was cross-contaminated during handling and storage. The efficiency of preconcentration and a preliminary identification was verified by the phenotyping technique after cultivation of the spores sampled from the apple surface.

Strategy to inactivate Clostridium perfringens spores in meat products.

PubMed

Akhtar, Saeed; Paredes-Sabja, Daniel; Torres, J Antonio; Sarker, Mahfuzur R

2009-05-01

The current study aimed to develop an inactivation strategy for Clostridium perfringens spores in meat through a combination of spore activation at low pressure (100-200 MPa, 7 min) and elevated temperature (80 degrees C, 10 min); spore germination at high temperatures (55, 60 or 65 degrees C); and inactivation of germinated spores with elevated temperatures (80 and 90 degrees C, 10 and 20 min) and high pressure (586 MPa, at 23 and 73 degrees C, 10 min). Low pressures (100-200 MPa) were insufficient to efficiently activate C. perfringens spores for germination. However, C. perfringens spores were efficiently activated with elevated temperature (80 degrees C, 10 min), and germinated at temperatures lethal for vegetative cells (>or= 55 degrees C) when incubated for 60 min with a mixture of L-asparagine and KCl (AK) in phosphate buffer (pH 7) and in poultry meat. Inactivation of spores (approximately 4 decimal reduction) in meat by elevated temperatures (80-90 degrees C for 20 min) required a long germination period (55 degrees C for 60 min). However, similar inactivation level was reached with shorter germination period (55 degrees C for 15 min) when spore contaminated-meat was treated with pressure-assisted thermal processing (568 MPa, 73 degrees C, 10 min). Therefore, the most efficient strategy to inactivate C. perfringens spores in poultry meat containing 50 mM AK consisted: (i) a primary heat treatment (80 degrees C, 10 min) to pasteurize and denature the meat proteins and to activate C. perfringens spores for germination; (ii) cooling of the product to 55 degrees C in about 20 min and further incubation at 55 degrees C for about 15 min for spore germination; and (iii) inactivation of germinated spores by pressure-assisted thermal processing (586 MPa at 73 degrees C for 10 min). Collectively, this study demonstrates the feasibility of an alternative and novel strategy to inactivate C. perfringens spores in meat products formulated with germinants specific for C. perfringens.

Reaerosolization of Fluidized Spores in Ventilation Systems▿

PubMed Central

Krauter, Paula; Biermann, Arthur

2007-01-01

This project examined dry, fluidized spore reaerosolization in a heating, ventilating, and air conditioning duct system. Experiments using spores of Bacillus atrophaeus, a nonpathogenic surrogate for Bacillus anthracis, were conducted to delineate the extent of spore reaerosolization behavior under normal indoor airflow conditions. Short-term (five air-volume exchanges), long-term (up to 21,000 air-volume exchanges), and cycled (on-off) reaerosolization tests were conducted using two common duct materials. Spores were released into the test apparatus in turbulent airflow (Reynolds number, 26,000). After the initial pulse of spores (approximately 1010 to 1011 viable spores) was released, high-efficiency particulate air filters were added to the air intake. Airflow was again used to perturb the spores that had previously deposited onto the duct. Resuspension rates on both steel and plastic duct materials were between 10−3 and 10−5 per second, which decreased to 10 times less than initial rates within 30 min. Pulsed flow caused an initial spike in spore resuspension concentration that rapidly decreased. The resuspension rates were greater than those predicted by resuspension models for contamination in the environment, a result attributed to surface roughness differences. There was no difference between spore reaerosolization from metal and that from plastic duct surfaces over 5 hours of constant airflow. The spores that deposited onto the duct remained a persistent source of contamination over a period of several hours. PMID:17293522

Protozoal Digestion of Coat-Defective Bacillus subtilis Spores Produces “Rinds” Composed of Insoluble Coat Protein▿

PubMed Central

Carroll, Alicia Monroe; Plomp, Marco; Malkin, Alexander J.; Setlow, Peter

2008-01-01

The Bacillus subtilis spore coat is a multilayer, proteinaceous structure that consists of more than 50 proteins. Located on the surface of the spore, the coat provides resistance to potentially toxic molecules as well as to predation by the protozoan Tetrahymena thermophila. When coat-defective spores are fed to Tetrahymena, the spores are readily digested. However, a residue termed a “rind” that looks like coat material remains. As observed with a phase-contrast microscope, the rinds are spherical or hemispherical structures that appear to be devoid of internal contents. Atomic force microscopy and chemical analyses showed that (i) the rinds are composed of insoluble protein largely derived from both outer and inner spore coat layers, (ii) the amorphous layer of the outer coat is largely responsible for providing spore resistance to protozoal digestion, and (iii) the rinds and intact spores do not contain significant levels of silicon. PMID:18689521

Bacillus subtilis spore survival and expression of germination-induced bioluminescence after prolonged incubation under simulated Mars atmospheric pressure and composition: implications for planetary protection and lithopanspermia

NASA Technical Reports Server (NTRS)

Nicholson, Wayne L.; Schuerger, Andrew C.

2005-01-01

Bacterial endospores in the genus Bacillus are considered good models for studying interplanetary transfer of microbes by natural or human processes. Although spore survival during transfer itself has been the subject of considerable study, the fate of spores in extraterrestrial environments has received less attention. In this report we subjected spores of a strain of Bacillus subtilis, containing luciferase resulting from expression of an sspB-luxAB gene fusion, to simulated martian atmospheric pressure (7-18 mbar) and composition (100% CO(2)) for up to 19 days in a Mars simulation chamber. We report here that survival was similar between spores exposed to Earth conditions and spores exposed up to 19 days to simulated martian conditions. However, germination-induced bioluminescence was lower in spores exposed to simulated martian atmosphere, which suggests sublethal impairment of some endogenous spore germination processes.

Microscopic Examination of Chitosan Polyphosphate Beads with Entrapped Spores of the Biocontrol Agent, Streptomyces melanosporofaciens EF-76

NASA Astrophysics Data System (ADS)

Jobin, Guy; Grondin, Gilles; Couture, Geneviève; Beaulieu, Carole

2005-04-01

Spores of the biocontrol agent, Streptomyces melanosporofaciens EF-76, were entrapped by complex coacervation in beads composed of a macromolecular complex (MC) of chitosan and polyphosphate. A proportion of spores entrapped in beads survived the entrapment procedure as shown by treating spores from chitosan beads with a dye allowing the differentiation of live and dead cells. The spore-loaded chitosan beads could be digested by a chitosanase, suggesting that, once introduced in soil, the beads would be degraded to release the biocontrol agent. Spore-loaded beads were examined by optical and scanning electron microscopy because the release of the biological agent depends on the spore distribution in the chitosan beads. The microscopic examination revealed that the beads had a porous surface and contained a network of inner microfibrils. Spores were entrapped in both the chitosan microfibrils and the bead lacuna.

Activation and injury of Clostridium perfringens spores by alcohols.

PubMed Central

Craven, S E; Blankenship, L C

1985-01-01

The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols. The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols. The response at a given temperature was dependent on the time of exposure and the alcohol concentration. The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C. The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character. Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation. Treatment with aqueous solutions of monohydric alcohols effectively activated C. perfringens spores and suggests a hydrophobic site for spore activation. PMID:2864897

Novel Fungitoxicity Assays for Inhibition of Germination-Associated Adhesion of Botrytis cinerea and Puccinia recondita Spores

PubMed Central

Slawecki, Richard A.; Ryan, Eileen P.; Young, David H.

2002-01-01

Botrytis cinerea and Puccinia recondita spores adhere strongly to polystyrene microtiter plates coincident with germination. We developed assays for inhibition of spore adhesion in 96-well microtiter plates by using sulforhodamine B staining to quantify the adherent spores. In both organisms, fungicides that inhibited germination strongly inhibited spore adhesion, with 50% effective concentrations (EC50s) comparable to those for inhibition of germination. In contrast, fungicides that acted after germination in B. cinerea inhibited spore adhesion to microtiter plates only at concentrations much higher than their EC50s for inhibition of mycelial growth. Similarly, in P. recondita the ergosterol biosynthesis inhibitors myclobutanil and fenbuconazole acted after germination and did not inhibit spore adhesion. The assays provide a rapid, high-throughput alternative to traditional spore germination assays and may be applicable to other fungi. PMID:11823196

Inactivation of Clostridium difficile spores by microwave irradiation.

PubMed

Ojha, Suvash Chandra; Chankhamhaengdecha, Surang; Singhakaew, Sombat; Ounjai, Puey; Janvilisri, Tavan

2016-04-01

Spores are a potent agent for Clostridium difficile transmission. Therefore, factors inhibiting spores have been of continued interest. In the present study, we investigated the influence of microwave irradiation in addition to conductive heating for C. difficile spore inactivation in aqueous suspension. The spores of 15 C. difficile isolates from different host origins were exposed to conductive heating and microwave irradiation. The complete inhibition of spore viability at 10(7) CFU/ml was encountered following microwave treatment at 800 W for 60 s, but was not observed in the conductive-heated spores at the same time-temperature exposure. The distinct patterns of ultrastructural alterations following microwave and conductive heat treatment were observed and the degree of damages by microwave was in the exposure time-dependent manner. Microwave would therefore be a simple and time-efficient tool to inactivate C. difficile spores, thus reducing the risk of C. difficile transmission. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fifth international fungus spore conference

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timberlake, W.E.

1993-04-01

This folio contains the proceedings of the Fifth International Fungal Spore Conference held August 17-21, 1991 at the Unicoi State Park at Helen, Georgia. The volume contains abstracts of each oral presentation as well as a collection of abstracts describing the poster sessions. Presentations were organized around the themes (1) Induction of Sporulation, (2) Nuclear Division, (3) Spore Formation, (4) Spore Release and Dispersal, and (4) Spore Germination.

Assessment of Resistance of Bacillus Horneckiae Endospores to UV Radiation and Function of Their Extraneous Layer in Resistance

NASA Technical Reports Server (NTRS)

Zachariah, Malcolm M.; Vaishampayan, Parag

2011-01-01

Spore-forming microbes are highly resistant to various physical and chemical conditions, which include ionizing and UV radiation, desiccation and oxidative stress, and the harsh environment of outer space or planetary surfaces. The spore's resistance might be due to their metabolically dormant state, and/or by the presence of a series of protective structures that encase the interior-most compartment, the core, which houses the spore chromosome. These spores have multiple layers surrounding the cell that are not found in vegetative cells, and some species have an outer layer of proteins and glycoproteins termed the "exosporium" or a fibrous "extraneous layer" (EL). Bacillus horneckiae is an EL-producing novel sporeformer isolated from a Phoenix spacecraft assembly clean room, and it has previously demonstrated resistance to UV radiation up to 1000 J/m(sup 2). The EL appears to bind B. horneckiae spores into large aggregations, or biofilms, and may confer some UV resistance to the spores. Multiple culturing and purification schemes were tried to achieve high purity spores because vegetative cells would skew UV resistance results. An ethanol-based purification scheme produced high purity spores. Selective removal of the EL from spores was attempted with two schemes: a chemical extraction method and physical extraction (sonication). Results from survival rates in the presence and absence of the external layer will provide a new understanding of the role of biofilms and passive resistance that may favor survival of biological systems in aggressive extra-terrestrial environments. The chemical extraction method decreased viable counts of spores and lead to an inconclusive change UV resistance relative to non-extracted spores. The physical extraction method lead to non-aggregated spores and did not alter viability; however, it produced UV resistance profiles similar to non-extracted spores. In addition to the EL-removal study, samples of B. horneckiae spores dried on aluminum coupons and exposed to increasing UV (200-400 nm range) levels (0 to 8.0 x 105 kJ/m(sup 2)) were tested for viability, which indicated that the maximum UV exposure level that still resulted in viable spores was 5.0 x 10? kJ/m(sup 2).

[Atmospheric concentration of fungus spores in Ankara and the effect of meteorological factors in 2003 period].

PubMed

Ceter, Talip; Pinar, Nur Münevver

2009-10-01

The atmospheric concentrations of airborne fungus spores change continuously according to the meteorological factors, and their intensity have important allergic effects on atopic subjects and opportunistic pathogenic effects on immunocompromised patients. The aim of this study was to identify the fungal spores found in Ankara atmosphere during 2003 period and to investigate the changes in spore concentrations in relation to meteorological factors. Fungal spores were sampled by using 7-day Burkard volumetric trap between January to December 2003, and probable identification was performed microscopically based on their morphological structures. A total of 433.079 spores/m3 belonging to 35 taxa were observed during the study. The rates of these taxa were as follows; 75.5% Cladosporium, 6.1% Alternaria, 2.2% Leptosphaeria, 2.2% Ustilago, 2.1% 1-septate ascospores, 2% Exosporium, 1.6% Pleospora, and 1.3% Drechslera. The other taxa with concentrations < 1% have consisted a total of 7.1% of all atmospheric spores (Puccinia, Curvularia, Coprinus, Nigrospora, Periconia, Melanomma, Torula, Ascobolus, Agrocybe, Pithomyces, Stemphyllium, Ganoderma, Boletus, Peronospora, Venturia, Paraphaeosphaeria, Epicoccum, Didymella, Chaetomium and Fusarium rates between 0.7-0.1%; Oidium, Xylaria, Botrytis, Melanospora, Dictyosporium, Sporormiella and Tetracoccosporium rates between 0.09-0.01%). Although fungal spores were detected in all months in Ankara atmosphere, the evaluation of the seasonal distribution of spore concentrations revealed that the highest value was detected in July (100.697 spores/m3), while the lowest value was in January (4268 spores/m3). When the effects of meteorological factors on spore concentrations were investigated, it was found that, monthly mean temperature (> 20 degrees C) has a strong positive correlation (p < 0.01), and monthly mean relative humidity (< %50) and precipitation (0-20 mm) have strong negative correlations (p < 0.01) on the spore concentrations, while wind velocity (3 m/s) has a slightly positive effect. An annual spore calendar which indicated weekly concentrations and allergenicity levels of those identified fungal spores, was also prepared in this study. In conclusion, it is expected that these data would be helpful for the researchers in the area of aeropalinology and for the clinicians to evaluate allergic diseases.

Inactivation of Clostridium perfringens spores adhered onto stainless steel surface by agents used in a clean-in-place procedure.

PubMed

Alzubeidi, Yasmeen S; Udompijitkul, Pathima; Talukdar, Prabhat K; Sarker, Mahfuzur R

2018-07-20

Enterotoxigenic Clostridium perfringens, a leading foodborne pathogen can be cross-contaminated from food processing stainless steel (SS) surfaces to the finished food products. This is mostly due to the high resistance of C. perfringens spores adhered onto SS surfaces to various disinfectants commonly used in food industries. In this study, we aimed to investigate the survivability and adherence of C. perfringens spores onto SS surfaces and then validate the effectiveness of a simulated Clean-in-Place (CIP) regime on inactivation of spores adhered onto SS surfaces. Our results demonstrated that, 1) C. perfringens spores adhered firmly onto SS surfaces and survived for at-least 48 h, unlike their vegetative cells who died within 30 min, after aerobic incubation at refrigerated and ambient temperatures; 2) Spores exhibited higher levels of hydrophobicity than vegetative cells, suggesting a correlation between cell surface hydrophobicity and adhesion to solid surfaces; 3) Intact spores were more hydrophobic than the decoated spores, suggesting a positive role of spore coat components on spores' hydrophobicity and thus adhesion onto SS surfaces; and finally 4) The CIP regime (NaOH + HNO 3 ) successfully inactivated C. perfringens spores adhered onto SS surfaces, and most of the effect of CIP regime appeared to be due to the NaOH. Collectively, our current findings may well contribute towards developing a strategy to control cross-contamination of C. perfringens spores into food products, which should help reducing the risk of C. perfringens-associated food poisoning outbreaks. Copyright © 2018 Elsevier B.V. All rights reserved.

Regulation and Characterization of a Newly Deduced Cell Wall Hydrolase Gene (cwlJ) Which Affects Germination of Bacillus subtilis Spores

PubMed Central

Ishikawa, Shu; Yamane, Kunio; Sekiguchi, Junichi

1998-01-01

The predicted amino acid sequence of Bacillus subtilis ycbQ (renamed cwlJ) exhibits high similarity to those of the deduced C-terminal catalytic domain of SleBs, the specific cortex-hydrolyzing enzyme of B. cereus and the deduced one of B. subtilis. We constructed a cwlJ::lacZ fusion in the B. subtilis chromosome. The β-galactosidase activity and results of Northern hybridization and primer extension analyses of the cwlJ gene indicated that it is transcribed by EςE RNA polymerase. cwlJ-deficient spores responded to both l-alanine and AGFK, the A580 values of spore suspensions decreased more slowly than in the case of the wild-type strain, and the mutant spores released less dipicolinic acid than did those of the wild-type strain during germination. However, the mutant spores released only slightly less hexosamine than did the wild-type spores. In contrast, B. subtilis sleB spores did not release hexosamine at a significant level. While cwlJ and sleB spores were able to germinate, CJSB (cwlJ sleB) spores could not germinate but exhibited initial germination reactions, e.g., partial decrease in A580 and slow release of dipicolinic acid. CJSB spores became slightly gray after 6 h in the germinant, but their refractility was much greater than that of sleB mutant spores. The roles of the sleB and cwlJ mutations in germination and spore maturation are also discussed. PMID:9515903

Assessing the Impact of Germination and Sporulation Conditions on the Adhesion of Bacillus Spores to Glass and Stainless Steel by Fluid Dynamic Gauging

PubMed Central

Xu Zhou, Ke; Li, Nan; Christie, Graham

2017-01-01

Abstract The adhesion of spores of 3 Bacillus species with distinctive morphologies to stainless steel and borosilicate glass was studied using the fluid dynamic gauging technique. Marked differences were observed between different species of spores, and also between spores of the same species prepared under different sporulation conditions. Spores of the food‐borne pathogen B. cereus were demonstrated to be capable of withstanding shear stresses greater than 1500 Pa when adhered to stainless steel, in contrast to spores of Bacillus subtilis and Bacillus megaterium, which detached in response to lower shear stress. An extended DLVO model was shown to be capable of predicting the relative differences in spore adhesion between spores of different species and different culture conditions, but did not predict absolute values of force of adhesion well. Applying the model to germinating spores showed a significant reduction in adhesion force shortly after triggering germination, indicating a potential strategy to achieve enhanced removal of spores from surfaces in response to shear stress, such as during cleaning‐in‐place procedures. Practical Application Spore‐forming bacteria are a concern to the food industry because they have the potential to cause food‐borne illness and product spoilage, while being strongly adhesive to processing surfaces and resistant to cleaning‐in‐place procedures. This work is of significance to the food processors and manufacturers because it offers insight to the properties of spore adhesion and identifies a potential strategy to facilitate the removal of spores during cleaning procedures. PMID:29125641

New ΦBT1 site-specific integrative vectors with neutral phenotype in Streptomyces.

PubMed

Gonzalez-Quiñonez, Nathaly; López-García, María Teresa; Yagüe, Paula; Rioseras, Beatriz; Pisciotta, Annalisa; Alduina, Rosa; Manteca, Ángel

2016-03-01

Integrative plasmids are one of the best options to introduce genes in low copy and in a stable form into bacteria. The ΦC31-derived plasmids constitute the most common integrative vectors used in Streptomyces. They integrate at different positions (attB and pseudo-attB sites) generating different mutations. The less common ΦBT1-derived vectors integrate at the unique attB site localized in the SCO4848 gene (S. coelicolor genome) or their orthologues in other streptomycetes. This work demonstrates that disruption of SCO4848 generates a delay in spore germination. SCO4848 is co-transcribed with SCO4849, and the spore germination phenotype is complemented by SCO4849. Plasmids pNG1-4 were created by modifying the ΦBT1 integrative vector pMS82 by introducing a copy of SCO4849 under the control of the promoter region of SCO4848. pNG2 and pNG4 also included a copy of the P ermE * in order to facilitate gene overexpression. pNG3 and pNG4 harboured a copy of the bla gene (ampicillin resistance) to facilitate selection in E. coli. pNG1-4 are the only integrative vectors designed to produce a neutral phenotype when they are integrated into the Streptomyces genome. The experimental approach developed in this work can be applied to create phenotypically neutral integrative plasmids in other bacteria.

Mushroom Emergence Detected by Combining Spore Trapping with Molecular Techniques.

PubMed

Castaño, Carles; Oliva, Jonàs; Martínez de Aragón, Juan; Alday, Josu G; Parladé, Javier; Pera, Joan; Bonet, José Antonio

2017-07-01

Obtaining reliable and representative mushroom production data requires time-consuming sampling schemes. In this paper, we assessed a simple methodology to detect mushroom emergence by trapping the fungal spores of the fruiting body community in plots where mushroom production was determined weekly. We compared the performance of filter paper traps with that of funnel traps and combined these spore trapping methods with species-specific quantitative real-time PCR and Illumina MiSeq to determine the spore abundance. Significantly more MiSeq proportional reads were generated for both ectomycorrhizal and saprotrophic fungal species using filter traps than were obtained using funnel traps. The spores of 37 fungal species that produced fruiting bodies in the study plots were identified. Spore community composition changed considerably over time due to the emergence of ephemeral fruiting bodies and rapid spore deposition (lasting from 1 to 2 weeks), which occurred in the absence of rainfall events. For many species, the emergence of epigeous fruiting bodies was followed by a peak in the relative abundance of their airborne spores. There were significant positive relationships between fruiting body yields and spore abundance in time for five of seven fungal species. There was no relationship between fruiting body yields and their spore abundance at plot level, indicating that some of the spores captured in each plot were arriving from the surrounding areas. Differences in fungal detection capacity by spore trapping may indicate different dispersal ability between fungal species. Further research can help to identify the spore rain patterns for most common fungal species. IMPORTANCE Mushroom monitoring represents a serious challenge in economic and logistical terms because sampling approaches demand extensive field work at both the spatial and temporal scales. In addition, the identification of fungal taxa depends on the expertise of experienced fungal taxonomists. Similarly, the study of fungal dispersal has been constrained by technological limitations, especially because the morphological identification of spores is a challenging and time-consuming task. Here, we demonstrate that spores from ectomycorrhizal and saprotrophic fungal species can be identified using simple spore traps together with either MiSeq fungus-specific amplicon sequencing or species-specific quantitative real-time PCR. In addition, the proposed methodology can be used to characterize the airborne fungal community and to detect mushroom emergence in forest ecosystems. Copyright © 2017 American Society for Microbiology.

Mushroom Emergence Detected by Combining Spore Trapping with Molecular Techniques

PubMed Central

Oliva, Jonàs; Martínez de Aragón, Juan; Alday, Josu G.; Parladé, Javier; Pera, Joan; Bonet, José Antonio

2017-01-01

ABSTRACT Obtaining reliable and representative mushroom production data requires time-consuming sampling schemes. In this paper, we assessed a simple methodology to detect mushroom emergence by trapping the fungal spores of the fruiting body community in plots where mushroom production was determined weekly. We compared the performance of filter paper traps with that of funnel traps and combined these spore trapping methods with species-specific quantitative real-time PCR and Illumina MiSeq to determine the spore abundance. Significantly more MiSeq proportional reads were generated for both ectomycorrhizal and saprotrophic fungal species using filter traps than were obtained using funnel traps. The spores of 37 fungal species that produced fruiting bodies in the study plots were identified. Spore community composition changed considerably over time due to the emergence of ephemeral fruiting bodies and rapid spore deposition (lasting from 1 to 2 weeks), which occurred in the absence of rainfall events. For many species, the emergence of epigeous fruiting bodies was followed by a peak in the relative abundance of their airborne spores. There were significant positive relationships between fruiting body yields and spore abundance in time for five of seven fungal species. There was no relationship between fruiting body yields and their spore abundance at plot level, indicating that some of the spores captured in each plot were arriving from the surrounding areas. Differences in fungal detection capacity by spore trapping may indicate different dispersal ability between fungal species. Further research can help to identify the spore rain patterns for most common fungal species. IMPORTANCE Mushroom monitoring represents a serious challenge in economic and logistical terms because sampling approaches demand extensive field work at both the spatial and temporal scales. In addition, the identification of fungal taxa depends on the expertise of experienced fungal taxonomists. Similarly, the study of fungal dispersal has been constrained by technological limitations, especially because the morphological identification of spores is a challenging and time-consuming task. Here, we demonstrate that spores from ectomycorrhizal and saprotrophic fungal species can be identified using simple spore traps together with either MiSeq fungus-specific amplicon sequencing or species-specific quantitative real-time PCR. In addition, the proposed methodology can be used to characterize the airborne fungal community and to detect mushroom emergence in forest ecosystems. PMID:28432095

Influence of food matrix on outgrowth heterogeneity of heat damaged Bacillus cereus spores.

PubMed

Warda, Alicja K; den Besten, Heidy M W; Sha, Na; Abee, Tjakko; Nierop Groot, Masja N

2015-05-18

Spoilage of heat treated foods can be caused by the presence of surviving spore-formers. It is virtually impossible to prevent contamination at the primary production level as spores are ubiquitous present in the environment and can contaminate raw products. As a result spore inactivation treatments are widely used by food producing industries to reduce the microbial spore loads. However consumers prefer mildly processed products that have less impact on its quality and this trend steers industry towards milder preservation treatments. Such treatments may result in damaged instead of inactivated spores, and these spores may germinate, repair, and grow out, possibly leading to quality and safety issues. The ability to repair and grow out is influenced by the properties of the food matrix. In the current communication we studied the outgrowth from heat damaged Bacillus cereus ATCC 14579 spores on Anopore membrane, which allowed following outgrowth heterogeneity of individual spores on broccoli and rice-based media as well as standard and mildly acidified (pH 5.5) meat-based BHI. Rice, broccoli and BHI pH 5.5 media resulted in delayed outgrowth from untreated spores, and increased heterogeneity compared to BHI pH 7.4, with the most pronounced effect in rice media. Exposure to wet heat for 1 min at 95 °C caused 2 log inactivation and approximately 95% of the spores in the surviving fraction were damaged resulting in substantial delay in outgrowth based on the time required to reach a maximum microcolony size of 256 cells. The delay was most pronounced for heat-treated spores on broccoli medium followed by spores on rice media (both untreated and treated). Interestingly, the increase in outgrowth heterogeneity of heat treated spores on BHI pH 7.4 was more pronounced than on rice, broccoli and BHI pH 5.5 conceivably reflecting that conditions in BHI pH 7.4 better support spore damage repair. This study compares the effects of three main factors, namely heat treatment, pH of BHI and the effect of food matrix highlighting the impact of different (model) food recovery media on outgrowth efficiency and heterogeneity of non-heat-treated and heat-damaged B. cereus spores. Copyright © 2015. Published by Elsevier B.V.

Inhibition of spore germination of Alternaria tenuis by sulfur dioxide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Couey, H.M.

1962-08-01

As a part of a continuing study of SO/sub 2/ fumigation of table grapes, the effect of SO/sub 2/ on spores of an isolate of A. tenuis Auct. causing decay of table grapes was determined. The amount of SO/sub 2/ required to inhibit completely spore germination depended on availability of moisture and the temperature. At 20/sup 0/C, wet spores required 20-min exposure to 100 ppm SO/sub 2/ to prevent germination, but spores equilibrated at 90% relative humidity (RH) required 10-min exposure to 1000 ppm SO/sub 2/. Dry spores at 60% RH were unaffected by a 20-min exposure to 4000 ppmmore » SO/sub 2/. Increasing the temperature in the range 5-20/sup 0/C increased effectiveness of the SO/sub 2/ treatment. A comparison of Alternaria with Botrytis cinerea Fr. (studied earlier) showed that wet spores of these organisms were about equally sensitive to SO/sub 2/, but that dry Alternaria spores were more resistant to SO/sub 2/ than dry Botrytis spores under comparable conditions.« less

Mushrooms as Rainmakers: How Spores Act as Nuclei for Raindrops

PubMed Central

2015-01-01

Millions of tons of fungal spores are dispersed in the atmosphere every year. These living cells, along with plant spores and pollen grains, may act as nuclei for condensation of water in clouds. Basidiospores released by mushrooms form a significant proportion of these aerosols, particularly above tropical forests. Mushroom spores are discharged from gills by the rapid displacement of a droplet of fluid on the cell surface. This droplet is formed by the condensation of water on the spore surface stimulated by the secretion of mannitol and other hygroscopic sugars. This fluid is carried with the spore during discharge, but evaporates once the spore is airborne. Using environmental electron microscopy, we have demonstrated that droplets reform on spores in humid air. The kinetics of this process suggest that basidiospores are especially effective as nuclei for the formation of large water drops in clouds. Through this mechanism, mushroom spores may promote rainfall in ecosystems that support large populations of ectomycorrhizal and saprotrophic basidiomycetes. Our research heightens interest in the global significance of the fungi and raises additional concerns about the sustainability of forests that depend on heavy precipitation. PMID:26509436

Calcineurin plays key roles in the dimorphic transition and virulence of the human pathogenic zygomycete Mucor circinelloides.

PubMed

Lee, Soo Chan; Li, Alicia; Calo, Silvia; Heitman, Joseph

2013-01-01

Many pathogenic fungi are dimorphic and switch between yeast and filamentous states. This switch alters host-microbe interactions and is critical for pathogenicity. However, in zygomycetes, whether dimorphism contributes to virulence is a central unanswered question. The pathogenic zygomycete Mucor circinelloides exhibits hyphal growth in aerobic conditions but switches to multi-budded yeast growth under anaerobic/high CO₂ conditions. We found that in the presence of the calcineurin inhibitor FK506, Mucor exhibits exclusively multi-budded yeast growth. We also found that M. circinelloides encodes three calcineurin catalytic A subunits (CnaA, CnaB, and CnaC) and one calcineurin regulatory B subunit (CnbR). Mutations in the latch region of CnbR and in the FKBP12-FK506 binding domain of CnaA result in hyphal growth of Mucor in the presence of FK506. Disruption of the cnbR gene encoding the sole calcineurin B subunit necessary for calcineurin activity yielded mutants locked in permanent yeast phase growth. These findings reveal that the calcineurin pathway plays key roles in the dimorphic transition from yeast to hyphae. The cnbR yeast-locked mutants are less virulent than the wild-type strain in a heterologous host system, providing evidence that hyphae or the yeast-hyphal transition are linked to virulence. Protein kinase A activity (PKA) is elevated during yeast growth under anaerobic conditions, in the presence of FK506, or in the yeast-locked cnbR mutants, suggesting a novel connection between PKA and calcineurin. cnaA mutants lacking the CnaA catalytic subunit are hypersensitive to calcineurin inhibitors, display a hyphal polarity defect, and produce a mixture of yeast and hyphae in aerobic culture. The cnaA mutants also produce spores that are larger than wild-type, and spore size is correlated with virulence potential. Our results demonstrate that the calcineurin pathway orchestrates the yeast-hyphal and spore size dimorphic transitions that contribute to virulence of this common zygomycete fungal pathogen.

Identifying the emerging human pathogen Scedosporium prolificans by using a species-specific monoclonal antibody that binds to the melanin biosynthetic enzyme tetrahydroxynaphthalene reductase.

PubMed

Thornton, Christopher R; Ryder, Lauren S; Le Cocq, Kate; Soanes, Darren M

2015-04-01

The dematiaceous (melanized) fungus Scedosporium prolificans is an emerging and frequently fatal pathogen of immunocompromised humans and which, along with the closely related fungi Pseudallescheria boydii, Scedosporium apiospermum and S. aurantiacum in the Pseudallescheria-Scedosporium complex, is a contributing aetiology to tsunami lung and central nervous system infections in near-drowning victims who have aspirated water laden with spores. At present, the natural habitat of the fungus is largely unknown, and accurate detection methods are needed to identify environmental reservoirs of infectious propagules. In this study, we report the development of a monoclonal antibody (mAb) (CA4) specific to S. prolificans, which does not cross-react with closely related fungi in the Pseudallescheria-Scedosporium complex or with a wide range of mould and yeast species pathogenic to humans. Using genome sequencing of a soil isolate and targeted gene disruption of the CA4 antigen-encoding gene, we show that mAb CA4 binds to the melanin-biosynthetic enzyme tetrahydroxynaphthalene reductase. Enzyme-deficient mutants produce orange-brown or green-brown spore suspensions compared with the black spore suspension of the wild-type strain. Using mAb CA4 and a mAb (HG12) specific to the related fungi P. boydii, P. apiosperma, S. apiospermum and S. aurantiacum, we demonstrate how the mAbs can be used in combination with a semiselective isolation procedure to track these opportunistic pathogens in environmental samples containing mixed populations of human pathogenic fungi. Specificity of mAb CA4 was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of fungi isolated from estuarine muds. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Anticarcinogenic effects of water extract of sporoderm-broken spores of Ganoderma lucidum on colorectal cancer in vitro and in vivo

PubMed Central

Na, Kun; Li, Kang; Sang, Tingting; Wu, Kaikai; Wang, Ying; Wang, Xingya

2017-01-01

Ganoderma lucidum (G. lucidum) polysaccharides (GLPs) have been used as traditional Chinese medicine for cancer prevention for many years. However, the mechanism by which GLP exerts its chemopreventive activities remains elusive. In addition, it is unclear whether sporoderm-broken spores of G. lucidum water extract (BSGLWE), which contains mainly GLPs, has anticancer effects on colorectal cancer. The present study investigated the anticancer effects and potential mechanisms of BSGLWE on colorectal cancer in vivo and in vitro. Our results showed that BSGLWE significantly inhibited colorectal cancer HCT116 cell viability in a time- and dose-dependent manner. Flow cytometry analysis indicated that BSGLWE disrupted cell cycle progression at G2/M phase via downregulation of cyclin B1 and cyclin A2, and upregulation of P21 at mRNA levels. Moreover, BSGLWE induced apoptosis by decreasing Bcl-2 and survivin at mRNA levels, and reduced Bcl-2, PARP, pro-caspase-3 and pro-caspase-9 at protein levels. Furthermore, BSGLWE suppressed tumor growth in vivo by regulating the expression of genes and proteins associated with cell cycle and apoptosis, which was further confirmed by a reduction of Ki67, PCNA, and Bcl-2 expression as determined by immunohistochemistry staining. NSAID activated gene-1 (NAG-1), a pro-apoptotic gene, was significantly upregulated in vivo and in vitro upon BSGLWE treatment at both mRNA and protein levels. In addition, the relative amounts of secreted NAG-1 in cell culture medium or serum of nude mice were all upregulated upon BSGLWE treatments, suggesting a role of NAG-1 in BSGLWE-induced anticolorectal cancer activity. This is the first study to show that BSGLWE inhibits colorectal cancer carcinogenesis through regulating genes responsible for cell proliferation, cell cycle and apoptosis cascades. These findings indicate that BSGLWE possesses chemopreventive potential in colorectal cancer which may serve as a promising anticancer agent for clinical applications. PMID:28358412

Pollen and spores as biological recorders of past ultraviolet irradiance.

NASA Astrophysics Data System (ADS)

Fraser, Wesley; Jardine, Phillip; Lomax, Barry; Sephton, Mark; Shanahan, Timothy; Miller, Charlotte; Gosling, William

2017-04-01

Ultraviolet (UV) irradiance from the Sun is a key driver of climatic and biotic change. UV irradiance modulates processes in the stratosphere, and influences the biosphere from ecosystem-level through to the largest scale patterns of diversification and extinction. Yet our understanding of UV irradiance is limited to the present; no validated empirical method exists to reconstruct UV flux over long, geologically relevant timescales. Here, we show that a recently developed proxy for UV irradiance based on spore and pollen chemistry can be used over long (100,000 years) timescales. First, we demonstrate spatial variation in spore and pollen chemistry correlate with known latitudinal solar irradiance gradients. Second, using this relationship we provide a reconstruction of past changes in solar irradiance based on the pollen record obtained from Lake Bosumtwi in Ghana. Variations in the chemistry of grass pollen from the Lake Bosumtwi record show a link to multiple orbital precessional cycles (19-21,000 years). By providing a unique, local proxy for broad spectrum solar irradiance, the chemical analysis of spores and pollen offers unprecedented opportunities to decouple solar variability, climate and vegetation change through geologic time and a new proxy with which to probe the Earth system.

Airborne pollen and spores of León (Spain)

NASA Astrophysics Data System (ADS)

Fernández-González, Delia; Suarez-Cervera, María; Díaz-González, Tomás; Valencia-Barrera, Rosa María

1993-06-01

A qualitative and quantitative analysis of airborne pollen and spores was carried out over 2 years (from September 1987 to August 1989) in the city of León. Slides were prepared daily using a volumetric pollen trap, which was placed on the Faculty of Veterinary Science building (University of León) 12m above ground-level. Fifty-one pollen types were observed; the most important of these were: Cupressaceae during the winter, Pinus and Quercus in spring, and Poaceae, Leguminosae and Chenopodiaceae in the summer. The results also showed the existence of a rich mould spore assemblage in the atmosphere. The group of Amerospores ( Penicillium, Aspergillus and Cladosporium) as well as Dictyospores ( Alternaria) were the most abundant; Puccinia was common in the air in August. Fluctuations in the total pollen and spores m3 of air were compared with meteorological parameters (temperature, relative humidity and rainfall). From the daily sampling of the atmosphere of León, considering the maximum and minimum temperature and duration of rainfall, the start of the pollen grain season was observed generally to coincide with a rise in temperature in the absence of rain.

The meiosis-specific nuclear passenger protein is required for proper assembly of forespore membrane in fission yeast.

PubMed

Takaine, Masak; Imada, Kazuki; Numata, Osamu; Nakamura, Taro; Nakano, Kentaro

2014-10-15

Sporulation, gametogenesis in yeast, consists of meiotic nuclear division and spore morphogenesis. In the fission yeast Schizosaccharomyces pombe, the four haploid nuclei produced after meiosis II are encapsulated by the forespore membrane (FSM), which is newly synthesized from spindle pole bodies (SPBs) in the cytoplasm of the mother cell as spore precursors. Although the coordination between meiosis and FSM assembly is vital for proper sporulation, the underlying mechanism remains unclear. In the present study, we identified a new meiosis-specific protein Npg1, and found that it was involved in the efficient formation of spores and spore viability. The accumulation and organization of the FSM was compromised in npg1-null cells, leading to the error-prone envelopment of nuclei. Npg1 was first seen as internuclear dots and translocated to the SPBs before the FSM assembled. Genetic analysis revealed that Npg1 worked in conjunction with the FSM proteins Spo3 and Meu14. These results suggest a possible signaling link from the nucleus to the meiotic SPBs in order to associate the onset of FSM assembly with meiosis II, which ensures the successful partitioning of gametic nuclei. © 2014. Published by The Company of Biologists Ltd.

Pollen and spores as biological recorders of past ultraviolet irradiance

NASA Astrophysics Data System (ADS)

Jardine, Phillip E.; Fraser, Wesley T.; Lomax, Barry H.; Sephton, Mark A.; Shanahan, Timothy M.; Miller, Charlotte S.; Gosling, William D.

2016-12-01

Solar ultraviolet (UV) irradiance is a key driver of climatic and biotic change. Ultraviolet irradiance modulates stratospheric warming and ozone production, and influences the biosphere from ecosystem-level processes through to the largest scale patterns of diversification and extinction. Yet our understanding of ultraviolet irradiance is limited because no method has been validated to reconstruct its flux over timescales relevant to climatic or biotic processes. Here, we show that a recently developed proxy for ultraviolet irradiance based on spore and pollen chemistry can be used over long (105 years) timescales. Firstly we demonstrate that spatial variations in spore and pollen chemistry correlate with known latitudinal solar irradiance gradients. Using this relationship we provide a reconstruction of past changes in solar irradiance based on the pollen record from Lake Bosumtwi in Ghana. As anticipated, variations in the chemistry of grass pollen from the Lake Bosumtwi record show a link to multiple orbital precessional cycles (19-21 thousand years). By providing a unique, local proxy for broad spectrum solar irradiance, the chemical analysis of spores and pollen offers unprecedented opportunities to decouple solar variability, climate and vegetation change through geologic time and a new proxy with which to probe the Earth system.

First insight into dead wood protistan diversity: a molecular sampling of bright-spored Myxomycetes (Amoebozoa, slime-moulds) in decaying beech logs.

PubMed

Clissmann, Fionn; Fiore-Donno, Anna Maria; Hoppe, Björn; Krüger, Dirk; Kahl, Tiemo; Unterseher, Martin; Schnittler, Martin

2015-06-01

Decaying wood hosts a large diversity of seldom investigated protists. Environmental sequencing offers novel insights into communities, but has rarely been applied to saproxylic protists. We investigated the diversity of bright-spored wood-inhabiting Myxomycetes by environmental sequencing. Myxomycetes have a complex life cycle culminating in the formation of mainly macroscopic fruiting bodies, highly variable in shape and colour that are often found on decaying logs. Our hypothesis was that diversity of bright-spored Myxomycetes would increase with decay. DNA was extracted from wood chips collected from 17 beech logs of varying decay stages from the Hainich-Dün region in Central Germany. We obtained 260 partial small subunit ribosomal RNA gene sequences of bright-spored Myxomycetes that were assembled into 29 OTUs, of which 65% were less than 98% similar to those in the existing database. The OTU richness revealed by molecular analysis surpassed that of a parallel inventory of fruiting bodies. We tested several environmental variables and identified pH, rather than decay stage, as the main structuring factor of myxomycete distribution. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Inhalation Anthrax: Dose Response and Risk Analysis

PubMed Central

Thran, Brandolyn; Morse, Stephen S.; Hugh-Jones, Martin; Massulik, Stacey

2008-01-01

The notion that inhalation of a single Bacillus anthracis spore is fatal has become entrenched nearly to the point of urban legend, in part because of incomplete articulation of the scientific basis for microbial risk assessment, particularly dose-response assessment. Risk analysis (ie, risk assessment, risk communication, risk management) necessitates transparency: distinguishing scientific facts, hypotheses, judgments, biases in interpretations, and potential misinformation. The difficulty in achieving transparency for biothreat risk is magnified by misinformation and poor characterization of both dose-response relationships and the driving mechanisms that cause susceptibility or resistance to disease progression. Regrettably, this entrenchment unnecessarily restricts preparedness planning to a single response scenario: decontaminate until no spores are detectable in air, water, or on surfaces—essentially forcing a zero-tolerance policy inconsistent with the biology of anthrax. We present evidence about inhalation anthrax dose-response relationships, including reports from multiple studies documenting exposures insufficient to cause inhalation anthrax in laboratory animals and humans. The emphasis of the article is clarification about what is known from objective scientific evidence for doses of anthrax spores associated with survival and mortality. From this knowledge base, we discuss the need for future applications of more formal risk analysis processes to guide development of alternative non-zero criteria or standards based on science to inform preparedness planning and other risk management activities. PMID:18582166

Classification of Streptomyces Spore Surfaces into Five Groups

PubMed Central

Dietz, Alma; Mathews, John

1971-01-01

Streptomyces spores surfaces have been classified into five groups, smooth, warty, spiny, hairy, and rugose, by examination of carbon replicas of spores with the transmission electron microscope and by direct examination of spores with the scanning electron microscope. Images PMID:4928607

Fifth international fungus spore conference. [Abstracts]: Final technical report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timberlake, W.E.

1993-04-01

This folio contains the proceedings of the Fifth International Fungal Spore Conference held August 17-21, 1991 at the Unicoi State Park at Helen, Georgia. The volume contains abstracts of each oral presentation as well as a collection of abstracts describing the poster sessions. Presentations were organized around the themes (1) Induction of Sporulation, (2) Nuclear Division, (3) Spore Formation, (4) Spore Release and Dispersal, and (4) Spore Germination.

Measuring Detachment of Aspergillus niger spores from Colonies with an Atomic Force Microscope.

PubMed

Li, Xian; Zhang, Tengfei Tim; Wang, Shugang

2018-06-26

Detachment of fungal spores from moldy surfaces and the subsequent aerosolization can lead to adverse health effects. Spore aerosolization occurs when the forces for aerosolization exceed the binding forces of spores with their colonies. The threshold force to detach a spore from a growing colony remains unknown. This investigation measured the detachment of spores of Aspergillus niger from a colony using an atomic force microscope (AFM). The spores were first affixed to the cantilever of the AFM with ultraviolet curing glue, and then the colony was moved downward until the spores detached. The threshold detachment forces were inferred from the deflection of the cantilever. In addition, the spores were aerosolized in a wind tunnel by a gradual increase of the blowing air speed. The forces measured by the AFM were compared with the hydrodynamic forces for aerosolization. The AFM measurements revealed that a force of 3.27 ± 0.25 nN was required to detach a single spore from the four-day-old colony, while 1.98 ± 0.13 nN was sufficient for the 10-day-old colony. Slightly smaller detachment forces were observed by the AFM than were determined by the aerosolization tests. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The Bacterial Endospore Stain on Schaeffer Fulton using Variation of Methylene Blue Solution

NASA Astrophysics Data System (ADS)

Oktari, A.; Supriatin, Y.; Kamal, M.; Syafrullah, H.

2017-02-01

Endospores staining is the type of staining to recognize the presence spore in bacterial vegetative cells. The bacterial endospores need a staining which can penetrate wall thickness of spore bacteria. A method of endospores staining is Schaeffer Fulton method that used Malachite Green. It is an alkaline substance staining that can staining the spore bacteria. In this research, it have found the alternative staining that can replace Malachite Green solution in spore bacterial stain. The alternative staining used is Methylene Blue solution (0,5 %, 0,7%, and 1% concentration) with pH variation (10, 11, and 12), and varyous heating time (3, 4, and 5 minutes). The all treatments staining have been effect on bacterial spores staining results. The warming time greatly affect the dye to penetrate the walls of bacterial spores, this can be seen in the results with various concentration at pH 10, indicates that the not long warm-up time 3 and 4 minutes, bacterial spores are not stained, while in the longer heating time is 5 minutes bacterial spores stained. This is caused because the longer heating time can make the pores of spore wall is open so that can facilitate the dye to get into the bacterial spores.

Test method development to evaluate hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and B. thuringiensis Al Hakam spores.

PubMed

Buhr, T L; Young, A A; Minter, Z A; Wells, C M; McPherson, D C; Hooban, C L; Johnson, C A; Prokop, E J; Crigler, J R

2012-11-01

To develop test methods and evaluate the survival of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air. Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH-adjusted bleach decontamination. Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores with similar kinetics. Hot, humid air is a potential alternative to conventional chemical decontamination. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Prediction of biological sensors appearance with ARIMA models as a tool for Integrated Pest Management protocols.

PubMed

Fernández-González, María; Ramos-Valcárcel, David; Aira, María Jesús; Rodríguez-Rajo, Francisco Javier

2016-01-01

Powdery mildew caused by Uncinula necator and Downy mildew produced by Plasmopara viticola are the most common diseases in the North-West Spain vineyards. Knowledge of airborne spore concentrations could be a useful tool in the Integrated Pest Management protocols in order to reduce the number of pesticide treatments, applied only when there is a real risk of infection. The study was carried out in a vineyard of the D. O. Ribeiro, in the North-West Spain, during the grapevine active period 2004-2012. A Hirts-type volumetric spore-trap was used for the aerobiological monitoring. During the study period the annual total U. necator spores amount ranged from the 578 spores registered in 2007 to the 4,145 spores sampled during 2008. The highest annual total P. viticola spores quantity was observed in 2010 (1,548 spores) and the lowest in 2005 (210 spores). In order to forecast the concentration of fungal spores, ARIMA models were elaborated. The most accurate models were an ARIMA (3.1.3) for U. necator and (1.0.3) for P. viticola. The possibility to forecast the spore presence 72 hours in advance open an important horizon for optimizing the organization of the harvest processes in the vineyard.

Fungal spores are transported long distances in smoke from biomass fires

NASA Astrophysics Data System (ADS)

Mims, Sarah A.; Mims, Forrest M.

Viable fungal spores are present in smoke from distant biomass fires. This finding has potentially important implications for prescribed burning, agricultural management and public health. While attempting to find fungal spores in dust blown from China to Texas, one of us (S.A.M.) discovered that smoke from Yucatan contains viable bacteria and fungal spores, including the genera Alternaria, Cladosporium, Fusariella and Curvularia. There was a high correlation ( r2=0.78) of spores and coarse carbon particles collected on microscope slides during 13 days of the 2002 smoke season. To eliminate possible contamination by local spores, an air sampler was flown from a kite at a Texas Gulf Coast beach during and after the 2003 smoke season on days when the NOAA back trajectory showed air arriving from Yucatan. Fifty-two spores and 19 coarse black carbon particles (>2.5 μm) were collected during a 30-min kite flight on the smoke day and 12 spores and four carbons on the day without smoke. We have found spores in smoke from an Arizona forest fire and in Asian smoke at Mauna Loa Observatory, Hawaii. We have tested these findings by burning dried grass, leaves, twigs and flood detritus. The smoke from all test fires contained many spores.

Comparison of Psilocybe cubensis spore and mycelium allergens.

PubMed

Helbling, A; Horner, W E; Lehrer, S B

1993-05-01

Basidiospores are an important cause of respiratory allergy in mold-sensitive atopic subjects. Collection of the large amounts of spores required for extract preparation is tedious and difficult. A desirable alternative could be mycelium grown in vitro if it is allergenically similar to spores. Therefore this study compared the allergen contents of Psilocybe cubensis spore and mycelium extracts by different techniques with the use of pooled sera from subjects who had skin test and RAST results that were positive to P. cubensis spores. Isoelectric focusing immunoprints revealed six common IgE-binding bands at isoelectric points 4.7, 5.0, 5.5, 5.6, 8.7, and 9.3. Two additional bands at isoelectric points 3.9 and 5.7 were detected only in the spore extract. Sodium dodecylsulfate-polyacrylamide gel electrophoresis immunoblots exhibited six common IgE-binding bands at 16, 35, 487, 52, 62, and 76 kd; 20 and 40 kd bands were present only in the spore extract. Although RAST and isoelectric focusing inhibition demonstrated that P. cubensis spore and mycelium extracts share many allergens, spores were allergenically more potent than mycelium. The results indicate that mycelium is a useful source of P. cubensis allergen, even though several spore allergens were not detected in mycelium.

The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation.

PubMed

Ribis, John W; Ravichandran, Priyanka; Putnam, Emily E; Pishdadian, Keyan; Shen, Aimee

2017-01-01

The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis , only two of these morphogenetic proteins have homologs in the Clostridia : SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis . Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis , C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia , but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis , indicating that this protein would not be a good target for inhibiting spore formation.

The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation

PubMed Central

Ribis, John W.; Ravichandran, Priyanka; Putnam, Emily E.; Pishdadian, Keyan

2017-01-01

ABSTRACT The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis, only two of these morphogenetic proteins have homologs in the Clostridia: SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis. Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis, C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia, but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis, indicating that this protein would not be a good target for inhibiting spore formation. PMID:28959733

Ultraviolet germicidal efficacy as a function of pulsed radiation parameters studied by spore film dosimetry.

PubMed

Bauer, Stefan; Holtschmidt, Hans; Ott, Günter

2018-01-01

Disinfection by pulsed ultraviolet (UV) radiation is a commonly used method, e.g. in industry or medicine and can be carried out either with lasers or broadband UV radiation sources. Detrimental effects to biological materials depending on parameters such as pulse duration τ or pulse repetition frequency f p are well-understood for pulsed coherent UV radiation, however, relatively little is known for its incoherent variant. Therefore, within this work, it is the first time that disinfection rates of pulsed and continuous (cw) incoherent UV radiation studied by means of spore film dosimetry are presented, compared with each other, and in a second step further investigated regarding two pulse parameters. After analyzing the dynamic range of the Bacillus subtilis spore films with variable cw radiant exposures H=5-100Jm -2 a validation of the Bunsen-Roscoe law revealed its restricted applicability and a 28% enhanced detrimental effect of pulsed compared to cw incoherent UV radiation. A radiant exposure H=50Jm -2 and an irradiance E=0.5Wm -2 were found to be suitable parameters for an analysis of the disinfection rate as a function of τ=0.5-10ms and f p =25-500Hz unveiling that shorter pulses and lower frequencies inactivate more spores. Finally, the number of applied pulses as well as the experiment time were considered with regard to spore film disinfection. Copyright © 2017 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woloshuk, C.P.; Kolattukudy, P.E.

Spores of the phytopathogenic fungus Fusarium solani f. sp. pisi were shown to produce the extracellular enzyme, cutinase, only when cutin or cutin hydrolysate was added to the spore suspension. Dihydroxy-C/sub 16/ acid and trihydroxy-C/sub 18/ acid, which are unique cutin monomers, showed the greatest cutinase-inducing activity. Experiments with several compounds structurally related to these fatty acids suggested that both a omega-hydroxyl and a midchain hydroxyl are required for cutinase-inducing activity. Cutinase appeared in the medium 30-45 min after the addition of the inducers to the spore suspension, and the activity level increased for 6 hr. Addition of cycloheximide (5more » ..mu..g/ml) completely inhibited cutinase production, suggesting that protein synthesis was involved in the increase of cutinase activity. Immunoblot analysis with rabbit antibodies prepared against cutinase showed that cutinase protein increased in parallel with the increase in enzyme activity. Measurement of cutinase-specific RNA levels by dot-blot hybridization with /sup 32/P-labeled cutinase cDNA showed that the cutinase gene transcripts could be detected within 15 min after addition of the inducers. Addition of exogenous cutinase greatly enhanced the level of cutinase gene transcripts induced by cutin. These results strongly suggest that the fungal spore senses that it is in contact with the plant by the production of small amounts of cutin monomers catalyzed by the low level of cutinase carried by the spore and that these monomers induce the synthesis of cutinase needed for penetration of the fungus into the plant.« less

Capability of the nematode-trapping fungus Duddingtonia flagrans to reduce infective larvae of gastrointestinal nematodes in goat feces in the southeastern United States: dose titration and dose time interval studies.

PubMed

Terrill, T H; Larsen, M; Samples, O; Husted, S; Miller, J E; Kaplan, R M; Gelaye, S

2004-04-15

Infection with gastrointestinal nematodes, particularly Haemonchus contortus, is a major constraint to goat production in the southeastern United States. Non-anthelmintic control alternatives are needed due to increasing resistance of these nematodes to available anthelmintics. Two studies were completed in Central Georgia in August 1999, and April-May 2000, using Spanish does naturally infected with Haemonchus contortus, Trichostongylus colubriformis, and Cooperia spp. to evaluate effectiveness of nematode-trapping fungi as a biological control agent. In the first experiment, five levels of Duddingtonia flagrans spores were mixed with a complete diet and fed once daily to the does (three per treatment) in metabolism crates. The treatment concentrations were (1) 5 x 10(5), (2) 2.5 x 10(5), (3) 10(5), and (4) 5 x 10(4) spores per kilogram body weight (BW), and (5) no spores. Fungal spores were fed for the first 7 days of the 14-day trial, and fecal samples were collected daily from individual animals for analysis of fecal egg count and establishment of fecal cultures. Efficacy of the fungus at reducing development of infective larvae (L3) in the fecal cultures was evaluated. The mean reduction in L3 from day 2 of the treatment period until the day after treatment stopped (days 2-8) was 93.6, 80.2, 84.1, and 60.8% for animals given the highest to lowest spore doses, respectively. Within 3-6 days after termination of fungal spore feedings, reduction in L3 development was no longer apparent in any of the treated animals. In a second experiment, effectiveness of 2.5 x 10(5) spores of D. flagrans per kilogram BW fed to does every day, every second day, and every third day was evaluated. Reduction in L3 development by daily feeding was less in the second experiment than in the first experiment. Daily fungal spore feeding provided more consistent larval reduction than intermittant feeding (every second or third day). When fed daily under controlled conditions, D. flagrans was effective in significantly reducing development of L3 and appears to be an effective tool for biocontrol of parasitic nematodes in goats.

Comparative analysis of the immunologic response induced by the Sterne 34F2 live spore Bacillus anthracis vaccine in a ruminant model.

PubMed

Ndumnego, Okechukwu C; Köhler, Susanne M; Crafford, Jannie; van Heerden, Henriette; Beyer, Wolfgang

2016-10-01

The Sterne 34F2 live spore vaccine (SLSV) developed in 1937 is the most widely used veterinary vaccine against anthrax. However, literature on the immunogenicity of this vaccine in a target ruminant host is scarce. In this study, we evaluated the humoral response to the Bacillus anthracis protective antigen (rPA), a recombinant bacillus collagen-like protein of anthracis (rBclA), formaldehyde inactivated spores (FIS) prepared from strain 34F2 and a vegetative antigen formulation prepared from a capsule and toxin deficient strain (CDC 1014) in Boer goats. The toxin neutralizing ability of induced antibodies was evaluated using an in vitro toxin neutralization assay. The protection afforded by the vaccine was also assessed in vaccinates. Anti-rPA, anti-FIS and lethal toxin neutralizing titres were superior after booster vaccinations, compared to single vaccinations. Qualitative analysis of humoral responses to rPA, rBclA and FIS antigens revealed a preponderance of anti-FIS IgG titres following either single or double vaccinations with the SLSV. Antibodies against FIS and rPA both increased by 350 and 300-fold following revaccinations respectively. There was no response to rBclA following vaccinations with the SLSV. Toxin neutralizing titres increased by 80-fold after single vaccination and 700-fold following a double vaccination. Lethal challenge studies in naïve goats indicated a minimum infective dose of 36 B. anthracis spores. Single and double vaccination with the SLSV protected 4/5 and 3/3 of goats challenged with>800 spores respectively. An early booster vaccination following the first immunization is suggested in order to achieve a robust immunity. Results from this study indicate that this crucial second vaccination can be administered as early as 3 months after the initial vaccination. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular and morphological characterisation of two species of the genus Ellipsomyxa Køie, 2003 (Ceratomyxidae) from the gall-bladder of Liza saliens (Risso) off Tunisian coasts of the Mediterranean.

PubMed

Thabet, Aouatef; Tlig-Zouari, Sabiha; Al Omar, Suliman Y; Mansour, Lamjed

2016-07-01

During examination of some species of the family Mugilidae, two coelozoic myxozoans were observed in the gall-bladder of the leaping mullet, Liza saliens (Risso). Spore morphology allowed us to allocate them to the genus Ellipsomyxa Køie, 2003, one of which is described here as new. Ellipsomyxa kalthoumi n. sp. was observed forming globular pseudoplasmodia and free spores floating in the bile. Mature spores are ellipsoidal, measuring 13-21 × 10-15 (17.2 × 13.2) µm and possessing two equal spherical polar capsules, 5-6 (5.5) µm in diameter, opening subterminally in opposite directions, with nine polar filament coils. Morphological data and molecular analysis of the small subunit rDNA sequences helped identify this parasite as a new species of Elliposmyxa. The second species identified as E. mugilis (Sitjà-Bobadilla & Alvarez-Pellitero, 1993) has oval spores with rounded ends, measuring 10-11 × 7-9 (10.5 × 8.0) µm and possessing two polar subspherical capsules, 2.7-3.0 (2.8) µm in diameter, opening subterminally in opposite directions. Spore morphometry and molecular study of the small subunit (SSU) of the rRNA gene identified this species as E. mugilis described from the same host in the Western Mediterranean off Spain. Phylogenetic analysis revealed Elliposmyxa as a monophyletic clade and showed that E. mugilis, E. syngnathi Køie & Karlsbakk, 2009, E. adlardi Whipps & Font, 2012 and E. gobii Køie, 2003 group in a subclade containing the Tunisian isolate of E. mugilis whereas E. kalthoumi n. sp. appears in a second subclade together with four Australian species, E. maniliensis Heiniger & Adlard, 2014, E. apogoni Heiniger & Adlard, 2014, E. nigropunctatis Heiniger & Adlard, 2014 and E. arothroni Heiniger & Adlard, 2014.

Seasonal Trends in Airborne Fungal Spores in Coastal California Ecosystems

NASA Astrophysics Data System (ADS)

Morfin, J.; Crandall, S. G.; Gilbert, G. S.

2014-12-01

Airborne fungal spores cause disease in plants and animals and may trigger respiratory illnesses in humans. In terrestrial systems, fungal sporulation, germination, and persistence are strongly regulated by local meteorological conditions. However, few studies investigate how microclimate affects the spatio-temporal dynamics of airborne spores. We measured fungal aerospora abundance and microclimate at varying spatial and time scales in coastal California in three habitat-types: coast redwood forest, mixed-evergreen forest, and maritime chaparral. We asked: 1) is there a difference in total airborne spore concentration between habitats, 2) when do we see peak spore counts, and 3) do spore densities correlate with microclimate conditions? Fungal spores were caught from the air with a volumetric vacuum air spore trap during the wet season (January - March) in 2013 and 2014, as well as monthly in 2014. Initial results suggest that mixed-evergreen forests exhibit the highest amounts of spore abundance in both years compared to the other habitats. This may be due to either a higher diversity of host plants in mixed-evergreen forests or a rich leaf litter layer that may harbor a greater abundance of saprotrophic fungi. Based on pilot data, we predict that temperature and to a lesser degree, relative humidity, will be important microclimate predictors for high spore densities. These data are important for understanding when and under what weather conditions we can expect to see high levels of fungal spores in the air; this can be useful information for managers who are interested in treating diseased plants with fungicides.

Bryophyte spore germinability is inhibited by peatland substrates

NASA Astrophysics Data System (ADS)

Bu, Zhao-Jun; Li, Zhi; Liu, Li-Jie; Sundberg, Sebastian; Feng, Ya-Min; Yang, Yun-He; Liu, Shuang; Song, Xue; Zhang, Xing-Lin

2017-01-01

Bryophyte substrates and species may affect spore germination through allelopathy. Polytrichum strictum is currently expanding in peatlands in north-eastern China - is this an effect of its superior spore germinability or do its gametophytes have a stronger allelopathic effect than do Sphagnum? We conducted a spore burial experiment to test the effect of species identity, substrate and water table depth (WTD) on spore germinability and bryophyte allelopathic effect with P. strictum and two Sphagnum species (S. palustre and S. magellanicum). After 5 months of burial during a growing season, the spores were tested for germinability. Allelopathic effect of bryophyte substrates was assessed by the difference between spore germinability after being stored inside or outside the substrates. After burial, more than 90% of the spores lost their germinability across all three species due to ageing and allelopathy. Spore germinability differed among species, where the spores in S. palustre had a higher germination frequency than those in P. strictum. The three bryophytes maintained a higher germinability in Sphagnum than in Polytrichum hummocks, probably due to a stronger allelopathic effect of P. strictum. Water table drawdown by 10 cm increased germinability by more than 60% across the three species. The study indicates that P. strictum does not possess an advantage regarding spore germination but rather its gametophytes have a stronger allelopathic effect. Due to the weaker inhibitive effect of Sphagnum gametophytes, P. strictum may have a potential establishment superiority over Sphagnum in peatlands, in addition to a better drought tolerance, which may explain its current expansion.

Arbuscular mycorrhizal fungal community composition associated with Juniperus brevifolia in native Azorean forest

NASA Astrophysics Data System (ADS)

Melo, Catarina Drumonde; Luna, Sara; Krüger, Claudia; Walker, Christopher; Mendonça, Duarte; Fonseca, Henrique M. A. C.; Jaizme-Vega, Maria; da Câmara Machado, Artur

2017-02-01

The communities of glomeromycotan fungi (arbuscular mycorrhizal fungi, AMF) under native Juniperus brevifolia forest from two Azorean islands, Terceira and São Miguel, were compared, mainly by spore morphology, and when possible, by molecular analysis. Thirty-nine morphotypes were detected from 12 genera. Glomeromycotan fungal richness was similar in Terceira and São Miguel, but significantly different among the four fragments of native forest. Spore diversity and community composition differed significantly between the two islands. The less degraded island, Terceira, showed 10 exclusive morphotypes including more rare types, whereas the more disturbed forest on São Miguel showed 13 morphs, mostly of common types. Forests from Terceira were dominated by Acaulosporaceae and Glomeraceae. Whereas members of Acaulosporaceae, Glomeraceae and Ambisporaceae were most frequent and abundant in those from São Miguel. Spore abundance was greatest on Terceira, and correlated with soil chemical properties (pH), average monthly temperature and relative humidity.

Standardization of Spore Inactivation Method for PMA-PhyloChip Analysis

NASA Technical Reports Server (NTRS)

Schrader, Michael

2011-01-01

In compliance with the Committee on Space Research (COSPAR) planetary protection policy, National Aeronautics and Space Administration (NASA) monitors the total microbial burden of spacecraft as a means for minimizing the inadvertent transfer of viable contaminant microorganisms to extraterrestrial environments (forward contamination). NASA standard assay-based counts are used both as a proxy for relative surface cleanliness and to estimate overall microbial burden as well as to assess whether forward planetary protection risk criteria are met for a given mission, which vary by the planetary body to be explored and whether or not life detection missions are present. Despite efforts to reduce presence of microorganisms from spacecraft prior to launch, microbes have been isolated from spacecraft and associated surfaces within the extreme conditions of clean room facilities using state of the art molecular technologies. Development of a more sensitive method that will better enumerate all viable microorganisms from spacecraft and associated surfaces could support future life detection missions. Current culture-based (NASA standard spore assay) and nucleic-acid-based polymerase chain reaction (PCR) methods have significant shortcomings in this type of analysis. The overall goal of this project is to evaluate and validate a new molecular method based on the use of a deoxyribonucleic acid (DNA) intercalating agent propidium monoazide (PMA). This is used in combination with DNA microarray (PhyloChip) which has been shown to identify very low levels of organisms on spacecraft associated surfaces. PMA can only penetrate the membrane of dead cells. Once penetrated, it intercalates the DNA and, upon photolysis using visible light it produces stable DNA monoadducts. This allows DNA to be unavailable for further PCR analysis. The specific aim of this study is to standardize the spore inactivation method for PMA-PhyloChip analysis. We have used the bacterial spores Bacillus subtilis 168 (standard laboratory isolate) as a test organism.

Comparative sensitivity to UV-B radiation of two Bacillus thuringiensis subspecies and other Bacillus sp.

PubMed

Myasnik, M; Manasherob, R; Ben-Dov, E; Zaritsky, A; Margalith, Y; Barak, Z

2001-08-01

Susceptibility of Bacillus thuringiensis spores and toxins to the UV-B range (280--330 nm) of the solar spectrum reaching Earth's surface may be responsible for its inactivation and low persistence in nature. Spores of the mosquito larvicidal B. thuringiensis subsp. israelensis were significantly more resistant to UV-B than spores of the lepidopteran-active subsp. kurstaki. Spores of subsp. israelensis were as resistant to UV-B as spores of B. subtilis and more resistant than spores of the closely related B. cereus and another mosquito larvicidal species B. sphaericus. Sensitivity of B. thuringiensis subsp. israelensis spores to UV-B radiation depended upon their culture age; 24-h cultures, approaching maximal larvicidal activity, were still sensitive. Maximal resistance to UV-B was achieved only at 48 h.

Cytological and proteomic analyses of horsetail (Equisetum arvense L.) spore germination

PubMed Central

Zhao, Qi; Gao, Jing; Suo, Jinwei; Chen, Sixue; Wang, Tai; Dai, Shaojun

2015-01-01

Spermatophyte pollen tubes and root hairs have been used as single-cell-type model systems to understand the molecular processes underlying polar growth of plant cells. Horsetail (Equisetum arvense L.) is a perennial herb species in Equisetopsida, which creates separately growing spring and summer stems in its life cycle. The mature chlorophyllous spores produced from spring stems can germinate without dormancy. Here we report the cellular features and protein expression patterns in five stages of horsetail spore germination (mature spores, rehydrated spores, double-celled spores, germinated spores, and spores with protonemal cells). Using 2-DE combined with mass spectrometry, 80 proteins were found to be abundance changed upon spore germination. Among them, proteins involved in photosynthesis, protein turnover, and energy supply were over-represented. Thirteen proteins appeared as proteoforms on the gels, indicating the potential importance of post-translational modification. In addition, the dynamic changes of ascorbate peroxidase, peroxiredoxin, and dehydroascorbate reductase implied that reactive oxygen species homeostasis is critical in regulating cell division and tip-growth. The time course of germination and diverse expression patterns of proteins in photosynthesis, energy supply, lipid and amino acid metabolism indicated that heterotrophic and autotrophic metabolism were necessary in light-dependent germination of the spores. Twenty-six proteins were involved in protein synthesis, folding, and degradation, indicating that protein turnover is vital to spore germination and rhizoid tip-growth. Furthermore, the altered abundance of 14-3-3 protein, small G protein Ran, actin, and caffeoyl-CoA O-methyltransferase revealed that signaling transduction, vesicle trafficking, cytoskeleton dynamics, and cell wall modulation were critical to cell division and polar growth. These findings lay a foundation toward understanding the molecular mechanisms underlying fern spore asymmetric division and rhizoid polar growth. PMID:26136760

Effects of culture conditions on the size, morphology and wet density of spores of Bacillus cereus 569 and Bacillus megaterium QM B1551.

PubMed

Xu Zhou, K; Wisnivesky, F; Wilson, D I; Christie, G

2017-07-01

The influence of variable culture conditions on the size and wet density of spores of Bacillus cereus and Bacillus megaterium were examined in this work. Culture temperature and initial pH was shown to have a significant impact on the size of both species, with increasingly alkaline culture media and elevated culture temperatures resulting in spores that were, on average, up to 25% reduced in volume. Increasing concentrations of inorganic salts in sporulation media exerted differing effects on each species; whereas a fivefold increase in the concentration of all salts resulted in only minor differences to the dimensions of B. cereus spores, B. megaterium spores became more elongated, displaying an average increase in volume of almost 30%. Similarly, as the spore elongated to yield aspect ratios larger than 1·4, their shape changed from typical prolate spheroids to cylinders with hemispherical ends. In contrast with previous studies, culture conditions employed in this study exerted no discernible impact on the wet density of B. cereus or B. megaterium spores. Bacterial spores of the genera Bacillus and Clostridium represent nature's most durable cells in terms of their extreme resistance to a variety of deleterious environments. As a result, they are of concern in the food processing, healthcare and other sectors, and are of increasing biotechnological interest. Improved understanding of variance in spore size, morphology and density may aid the development of certain spore-associated applications (e.g. spore surface display) while contributing to active areas of research such as spore adhesion and resistance to heat. © 2017 The Authors. Letters in Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.

Decontamination Options for Bacillus anthracis-Contaminated Drinking Water Determined from Spore Surrogate Studies ▿

PubMed Central

Raber, Ellen; Burklund, Alison

2010-01-01

Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination alternatives for use in a contaminated drinking water supply. The parameters were as follows: (i) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus), (ii) spore concentration in suspension (102 and 106 spores/ml), (iii) chemical characteristics of the decontaminant (sodium dichloro-S-triazinetrione dihydrate [Dichlor], hydrogen peroxide, potassium peroxymonosulfate [Oxone], sodium hypochlorite, and VirkonS), (iv) decontaminant concentration (0.01% to 5%), and (v) exposure time to decontaminant (10 min to 1 h). Results from 138 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5% and Dichlor or sodium hypochlorite at a concentration of 2% were highly effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor's desirable characteristics of high oxidation potential, high level of free chlorine, and a more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting the EPA biocide standard of greater than a 6-log kill after a 10-min exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS and Oxone were less effective as decontaminants than other options evaluated in this study and did not meet the EPA's efficacy standard for a biocide, although they were found to be as effective for concentrations of 102 spores/ml. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult. PMID:20709855

Airborne fungal spores of Alternaria, meteorological parameters and predicting variables

NASA Astrophysics Data System (ADS)

Filali Ben Sidel, Farah; Bouziane, Hassan; del Mar Trigo, Maria; El Haskouri, Fatima; Bardei, Fadoua; Redouane, Abdelbari; Kadiri, Mohamed; Riadi, Hassane; Kazzaz, Mohamed

2015-03-01

Alternaria is frequently found as airborne fungal spores and is recognized as an important cause of respiratory allergies. The aerobiological monitoring of fungal spores was performed using a Burkard volumetric spore traps. To establish predicting variables for daily and weakly spore counts, a stepwise multiple regression between spore concentrations and independent variables (meteorological parameters and lagged values from the series of spore concentrations: previous day or week concentration (Alt t - 1) and mean concentration of the same day or week in other years ( C mean)) was made with data obtained during 2009-2011. Alternaria conidia are present throughout the year in the atmosphere of Tetouan, although they show important seasonal fluctuations. The highest levels of Alternaria spores were recorded during the spring and summer or autumn. Alternaria showed maximum daily values in April, May or October depending on year. When the spore variables of Alternaria, namely C mean and Alt t - 1, and meteorological parameters were included in the equation, the resulting R 2 satisfactorily predict future concentrations for 55.5 to 81.6 % during the main spore season and the pre-peak 2. In the predictive model using weekly values, the adjusted R 2 varied from 0.655 to 0.676. The Wilcoxon test was used to compare the results from the expected values and the pre-peak spore data or weekly values for 2012, indicating that there were no significant differences between series compared. This test showed the C mean, Alt t - 1, frequency of the wind third quadrant, maximum wind speed and minimum relative humidity as the most efficient independent variables to forecast the overall trend of this spore in the air.

Model simulations of fungal spore distribution over the Indian region

NASA Astrophysics Data System (ADS)

Ansari, Tabish U.; Valsan, Aswathy E.; Ojha, N.; Ravikrishna, R.; Narasimhan, Balaji; Gunthe, Sachin S.

2015-12-01

Fungal spores play important role in the health of humans, animals, and plants by constituting a class of the primary biological aerosol particles (PBAPs). Additionally, these could mediate the hydrological cycle by acting as nuclei for ice and cloud formation (IN and CCN respectively). Various processes in the biosphere and the variations in the meteorological conditions control the releasing mechanism of spores through active wet and dry discharge. In the present paper, we simulate the concentration of fungal spores over the Indian region during three distinct meteorological seasons by combining a numerical model (WRF-Chem) with the fungal spore emissions based on land-use type. Maiden high-resolution regional simulations revealed large spatial gradient and strong seasonal dependence in the concentration of fungal spores over the Indian region. The fungal spore concentrations are found to be the highest during winter (0-70 μg m-3 in December), moderately higher during summer (0-35 μg m-3 in May) and lowest during the monsoon (0-25 μg m-3 in July). The elevated concentrations during winter are attributed to the shallower boundary layer trapping the emitted fungal spores in smaller volume. In contrast, the deeper boundary layer mixing in May and stronger monsoonal-convection in July distribute the fungal spores throughout the lower troposphere (∼5 km). We suggest that the higher fungal spore concentrations during winter could have potential health impacts. While, stronger vertical mixing could enable fungal spores to influence the cloud formation during summer and monsoon. Our study provides the first information about the distribution and seasonal variation of fungal spores over the densely populated and observationally sparse Indian region.

Elastic and inelastic light scattering from single bacterial spores in an optical trap allows the monitoring of spore germination dynamics

PubMed Central

Peng, Lixin; Chen, De; Setlow, Peter; Li, Yong-qing

2009-01-01

Raman scattering spectroscopy and elastic light scattering intensity (ESLI) were used to simultaneously measure levels of Ca-dipicolinic acid (CaDPA) and changes in spore morphology and refractive index during germination of individual B. subtilis spores with and without the two redundant enzymes (CLEs), CwlJ and SleB, that degrade spores’ peptidoglycan cortex. Conclusions from these measurements include: 1) CaDPA release from individual wild-type germinating spores was biphasic; in a first heterogeneous slow phase, Tlag, CaDPA levels decreased ∼15% and in the second phase ending at Trelease, remaining CaDPA was released rapidly; 2) in L-alanine germination of wild-type spores and spores lacking SleB: a) the ESLI rose ∼2-fold shortly before Tlag at T1; b) following Tlag, the ESLI again rose ∼2-fold at T2 when CaDPA levels had decreased ∼50%; and c) the ESLI reached its maximum value at ∼Trelease and then decreased; 3) in CaDPA germination of wild-type spores: a) Tlag increased and the first increase in ESLI occurred well before Tlag, consistent with different pathways for CaDPA and L-alanine germination; b) at Trelease the ESLI again reached its maximum value; 4) in L-alanine germination of spores lacking both CLEs and unable to degrade their cortex, the time ΔTrelease (Trelease–Tlag) for excretion of ≥75% of CaDPA was ∼15-fold higher than that for wild-type or sleB spores; and 5) spores lacking only CwlJ exhibited a similar, but not identical ESLI pattern during L-alanine germination to that seen with cwlJ sleB spores, and the high value for ΔTrelease. PMID:19374431

Germination of Spores of Astrobiologically Relevant Bacillus Species in High-Salinity Environments.

PubMed

Nagler, Katja; Julius, Christina; Moeller, Ralf

2016-07-01

In times of increasing space exploration and search for extraterrestrial life, new questions and challenges for planetary protection, aiming to avoid forward contamination of different planets or moons with terrestrial life, are emerging. Spore-forming bacteria such as Bacillus species have a high contamination potential due to their spores' extreme resistance, enabling them to withstand space conditions. Spores require liquid water for their conversion into a growing cell (i.e., spore germination and subsequent growth). If present, water on extraterrestrial planets or moons is likely to be closely associated with salts (e.g., in salty oceans or brines), thus constituting high-salinity environments. Spores of Bacillus subtilis can germinate despite very high salt concentrations, although salt stress does exert negative effects on this process. In this study, germination and metabolic reactivation ("outgrowth") of spores of five astrobiologically relevant Bacillus species (B. megaterium, B. pumilus SAFR-032, B. nealsonii, B. mojavensis, and B. vallismortis) in high salinity (≤3.6 M NaCl) were investigated. Spores of different species exhibited different germination and outgrowth capabilities in high salinity, which strongly depended on germination conditions, especially the exact composition of the medium. In this context, a new "universal" germination trigger for Bacillus spores, named KAGE (KCl, L-alanine, D-glucose, ectoine), was identified, which will be very useful for future comparative germination and outgrowth studies on different Bacillus species. Overall, this study yielded interesting new insights on salt stress effects on spore germination and points out the difficulty of predicting the potential of spores to contaminate salty environments on extraterrestrial celestial bodies. Bacillus species-Spores-Germination-High salinity-Salt stress-NaCl-Inhibition. Astrobiology 16, 500-512.

Moist-Heat Resistance, Spore Aging, and Superdormancy in Clostridium difficile▿†

PubMed Central

Rodriguez-Palacios, Alexander; LeJeune, Jeffrey T.

2011-01-01

Clostridium difficile spores can survive extended heating at 71°C (160°F), a minimum temperature commonly recommended for adequate cooking of meats. To determine the extent to which higher temperatures would be more effective at killing C. difficile, we quantified (D values) the effect of moist heat at 85°C (145°F, for 0 to 30 min) on C. difficile spores and compared it to the effects at 71 and 63°C. Fresh (1-week-old) and aged (≥20-week-old) C. difficile spores from food and food animals were tested in multiple experiments. Heating at 85°C markedly reduced spore recovery in all experiments (5 to 6 log10 within 15 min of heating; P < 0.001), regardless of spore age. In ground beef, the inhibitory effect of 85°C was also reproducible (P < 0.001), but heating at 96°C reduced 6 log10 within 1 to 2 min. Mechanistically, optical density and enumeration experiments indicated that 85°C inhibits cell division but not germination, but the inhibitory effect was reversible in some spores. Heating at 63°C reduced counts for fresh spores (1 log10, 30 min; P < 0.04) but increased counts of 20-week-old spores by 30% (15 min; P < 0.02), indicating that sublethal heat treatment reactivates superdormant spores. Superdormancy is an increasingly recognized characteristic in Bacillus spp., and it is likely to occur in C. difficile as spores age. The potential for reactivation of (super)dormant spores with sublethal temperatures may be a food safety concern, but it also has potential diagnostic value. Ensuring that food is heated to >85°C would be a simple and important intervention to reduce the risk of inadvertent ingestion of C. difficile spores. PMID:21398481

Novel Strategies for Enhanced Removal of Persistent Bacillus anthracis Surrogates and Clostridium difficile Spores from Skin

PubMed Central

Nerandzic, Michelle M.; Rackaityte, Elze; Jury, Lucy A.; Eckart, Kevin; Donskey, Curtis J.

2013-01-01

Background Removing spores of Clostridium difficile and Bacillus anthracis from skin is challenging because they are resistant to commonly used antimicrobials and soap and water washing provides only modest efficacy. We hypothesized that hygiene interventions incorporating a sporicidal electrochemically generated hypochlorous acid solution (Vashe®) would reduce the burden of spores on skin. Methods Hands of volunteers were inoculated with non-toxigenic C. difficile spores or B. anthracis spore surrogates to assess the effectiveness of Vashe solution for reducing spores on skin. Reduction in spores was compared for Vashe hygiene interventions versus soap and water (control). To determine the effectiveness of Vashe solution for removal of C. difficile spores from the skin of patients with C. difficile infection (CDI), reductions in levels of spores on skin were compared for soap and water versus Vashe bed baths. Results Spore removal from hands was enhanced with Vashe soak (>2.5 log10 reduction) versus soap and water wash or soak (~2.0 log10 reduction; P <0.05) and Vashe wipes versus alcohol wipes (P <0.01). A combined approach of soap and water wash followed by soaking in Vashe removed >3.5 log10 spores from hands (P <0.01 compared to washing or soaking alone). Bed baths using soap and water (N =26 patients) did not reduce the percentage of positive skin cultures for CDI patients (64% before versus 57% after bathing; P =0.5), whereas bathing with Vashe solution (N =21 patients) significantly reduced skin contamination (54% before versus 8% after bathing; P =0.0001). Vashe was well-tolerated with no evidence of adverse effects on skin. Conclusions Vashe was safe and effective for reducing the burden of B. anthracis surrogates and C. difficile spores on hands. Bed baths with Vashe were effective for reducing C. difficile on skin. These findings suggest a novel strategy to reduce the burden of spores on skin. PMID:23844234

Surface display of bacterial tyrosinase on spores of Bacillus subtilis using CotE as an anchor protein.

PubMed

Hosseini-Abari, Afrouzossadat; Kim, Byung-Gee; Lee, Sang-Hyuk; Emtiazi, Giti; Kim, Wooil; Kim, June-Hyung

2016-12-01

Tyrosinases, copper-containing monooxygenases, are widely used enzymes for industrial, medical, and environmental applications. We report the first functional surface display of Bacillus megaterium tyrosinase on Bacillus subtilis spores using CotE as an anchor protein. Flow Cytometry was used to verify surface expression of tyrosinase on the purified spores. Moreover, tyrosinase activity of the displayed enzyme on B. subtilis spores was monitored in the presence of L-tyrosine (substrate) and CuSO 4 (inducer). The stability of the spore-displayed tyrosinase was then evaluated after 15 days maintenance of the spores at room temperature, and no significant decrease in the enzyme activity was observed. In addition, the tyrosinase-expressing spores could be repeatedly used with 62% retained enzymatic activity after six times washing with Tris-HCl buffer. This genetically immobilized tyrosinase on the spores would make a new advance in industrial, medical, and environmental applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Response surface modeling for hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores

PubMed Central

2014-01-01

Response surface methodology using a face-centered cube design was used to describe and predict spore inactivation of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure of six spore-contaminated materials to hot, humid air. For each strain/material pair, an attempt was made to fit a first or second order model. All three independent predictor variables (temperature, relative humidity, and time) were significant in the models except that time was not significant for B. thuringiensis Al Hakam on nylon. Modeling was unsuccessful for wiring insulation and wet spores because there was complete spore inactivation in the majority of the experimental space. In cases where a predictive equation could be fit, response surface plots with time set to four days were generated. The survival of highly purified Bacillus spores can be predicted for most materials tested when given the settings for temperature, relative humidity, and time. These predictions were cross-checked with spore inactivation measurements. PMID:24949256

Differentiation of Dictyostelium discoideum vegetative cells into spores during earth orbit in space

NASA Astrophysics Data System (ADS)

Takahashi, A.; Ohnishi, K.; Takahashi, S.; Masukawa, M.; Sekikawa, K.; Amano, T.; Nakano, T.; Nagaoka, S.; Ohnishi, T.

2001-01-01

We reported previously that emerged amoebae of Dictyosterium ( D.) discoideum grew, aggregated and differentiated to fruiting bodies with normal morphology in space. Here, we investigated the effects of space radiation and/or microgravity on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space in a radiation-sensitive strain, γs13, and the parental strain, NC4. In γs13, there were hardly spores in the fruiting bodies formed in space. In NC4, we found a decrease in the number of spores, a delay in germination of the spores and delayed start of cell growth of the spores formed in space when compared to the ground control. However, the mutation frequency of the NC4 spores formed in space was similar to that of the ground control. We conclude that the depression of spore formation might be induced by microgravity and/or space radiation through the depression of some stage(s) of DNA repair during cell differentiation in the slime mold.

Spore coat architecture of Clostridium novyi NT spores.

PubMed

Plomp, Marco; McCaffery, J Michael; Cheong, Ian; Huang, Xin; Bettegowda, Chetan; Kinzler, Kenneth W; Zhou, Shibin; Vogelstein, Bert; Malkin, Alexander J

2007-09-01

Spores of the anaerobic bacterium Clostridium novyi NT are able to germinate in and destroy hypoxic regions of tumors in experimental animals. Future progress in this area will benefit from a better understanding of the germination and outgrowth processes that are essential for the tumorilytic properties of these spores. Toward this end, we have used both transmission electron microscopy and atomic force microscopy to determine the structure of both dormant and germinating spores. We found that the spores are surrounded by an amorphous layer intertwined with honeycomb parasporal layers. Moreover, the spore coat layers had apparently self-assembled, and this assembly was likely to be governed by crystal growth principles. During germination and outgrowth, the honeycomb layers, as well as the underlying spore coat and undercoat layers, sequentially dissolved until the vegetative cell was released. In addition to their implications for understanding the biology of C. novyi NT, these studies document the presence of proteinaceous growth spirals in a biological organism.

Dual effects of single-walled carbon nanotubes coupled with near-infrared radiation on Bacillus anthracis spores: inactivates spores and stimulates the germination of surviving spores

PubMed Central

2013-01-01

Background Bacillus anthracis is a pathogen that causes life-threatening disease--anthrax. B. anthracis spores are highly resistant to extreme temperatures and harsh chemicals. Inactivation of B. anthracis spores is important to ensure the environmental safety and public health. The 2001 bioterrorism attack involving anthrax spores has brought acute public attention and triggered extensive research on inactivation of B. anthracis spores. Single-walled carbon nanotubes (SWCNTs) as a class of emerging nanomaterial have been reported as a strong antimicrobial agent. In addition, continuous near infrared (NIR) radiation on SWCNTs induces excessive local heating which can enhance SWCNTs’ antimicrobial effect. In this study, we investigated the effects of SWCNTs coupled with NIR treatment on Bacillus anthracis spores. Results and discussion The results showed that the treatment of 10 μg/mL SWCNTs coupled with 20 min NIR significantly improved the antimicrobial effect by doubling the percentage of viable spore number reduction compared with SWCNTs alone treatment (88% vs. 42%). At the same time, SWCNTs-NIR treatment activated the germination of surviving spores and their dipicolinic acid (DPA) release during germination. The results suggested the dual effect of SWCNTs-NIR treatment on B. anthracis spores: enhanced the sporicidal effect and stimulated the germination of surviving spores. Molecular level examination showed that SWCNTs-NIR increased the expression levels (>2-fold) in 3 out of 6 germination related genes tested in this study, which was correlated to the activated germination and DPA release. SWCNTs-NIR treatment either induced or inhibited the expression of 3 regulatory genes detected in this study. When the NIR treatment time was 5 or 25 min, there were 3 out of 7 virulence related genes that showed significant decrease on expression levels (>2 fold decrease). Conclusions The results of this study demonstrated the dual effect of SWCNTs-NIR treatment on B. anthracis spores, which enhanced the sporicidal effect and stimulated the germination of surviving spores. SWCNTs-NIR treatment also altered the expression of germination, regulatory, and virulence-related genes in B. anthracis. PMID:23965258

Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes.

PubMed

Mattossovich, Rosanna; Iacono, Roberta; Cangiano, Giuseppina; Cobucci-Ponzano, Beatrice; Isticato, Rachele; Moracci, Marco; Ricca, Ezio

2017-11-28

The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-D-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-D-xylans to remove successive D-xylose residues from the non-reducing termini. We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation. Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes for single as well as multi-step reactions.

Characterization of Wet-Heat Inactivation of Single Spores of Bacillus Species by Dual-Trap Raman Spectroscopy and Elastic Light Scattering▿

PubMed Central

Zhang, Pengfei; Kong, Lingbo; Setlow, Peter; Li, Yong-qing

2010-01-01

Dual-trap laser tweezers Raman spectroscopy (LTRS) and elastic light scattering (ELS) were used to investigate dynamic processes during high-temperature treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis in water. Major conclusions from these studies included the following. (i) After spores of all three species were added to water at 80 to 90°C, the level of the 1:1 complex of Ca2+ and dipicolinic acid (CaDPA; ∼25% of the dry weight of the spore core) in individual spores remained relatively constant during a highly variable lag time (Tlag), and then CaDPA was released within 1 to 2 min. (ii) The Tlag values prior to rapid CaDPA release and thus the times for wet-heat killing of individual spores of all three species were very heterogeneous. (iii) The heterogeneity in kinetics of wet-heat killing of individual spores was not due to differences in the microscopic physical environments during heat treatment. (iv) During the wet-heat treatment of spores of all three species, spore protein denaturation largely but not completely accompanied rapid CaDPA release, as some changes in protein structure preceded rapid CaDPA release. (v) Changes in the ELS from individual spores of all three species were strongly correlated with the release of CaDPA. The ELS intensities of B. cereus and B. megaterium spores decreased gradually and reached minima at T1 when ∼80% of spore CaDPA was released, then increased rapidly until T2 when full CaDPA release was complete, and then remained nearly constant. The ELS intensity of B. subtilis spores showed similar features, although the intensity changed minimally, if at all, prior to T1. (vi) Carotenoids in B. megaterium spores' inner membranes exhibited two changes during heat treatment. First, the carotenoid's two Raman bands at 1,155 and 1,516 cm−1 decreased rapidly to a low value and to zero, respectively, well before Tlag, and then the residual 1,155-cm−1 band disappeared, in parallel with the rapid CaDPA release beginning at Tlag. PMID:20097820

Binding Affinity of Glycoconjugates to BACILLUS Spores and Toxins

NASA Astrophysics Data System (ADS)

Rasol, Aveen; Eassa, Souzan; Tarasenko, Olga

2010-04-01

Early recognition of Bacillus cereus group species is important since they can cause food-borne illnesses and deadly diseases in humans. Glycoconjugates (GCs) are carbohydrates covalently linked to non-sugar moieties including lipids, proteins or other entities. GCs are involved in recognition and signaling processes intrinsic to biochemical functions in cells. They also stimulate cell-cell adhesion and subsequent recognition and activation of receptors. We have demonstrated that GCs are involved in Bacillus cereus spore recognition. In the present study, we have investigated whether GCs possess the ability to bind and recognize B. cereus spores and Bacillus anthracis recombinant single toxins (sTX) and complex toxins (cTX). The affinity of GCs to spores + sTX and spores + cTX toxins was studied in the binding essay. Our results demonstrated that GC9 and GC10 were able to selectively bind to B. cereus spores and B. anthracis toxins. Different binding affinities for GCs were found toward Bacillus cereus spores + sTX and spores + cTX. Dilution of GCs does not impede the recognition and binding. Developed method provides a tool for simultaneous recognition and targeting of spores, bacteria toxins, and/or other entities.

Rapid detection of Bacillus anthracis spores using a super-paramagnetic lateral-flow immunological detection system.

PubMed

Wang, Dian-Bing; Tian, Bo; Zhang, Zhi-Ping; Deng, Jiao-Yu; Cui, Zong-Qiang; Yang, Rui-Fu; Wang, Xu-Ying; Wei, Hong-Ping; Zhang, Xian-En

2013-04-15

There is an urgent need for convenient, sensitive, and specific methods to detect the spores of Bacillus anthracis, the causative agent of anthrax, because of the bioterrorism threat posed by this bacterium. In this study, we firstly develop a super-paramagnetic lateral-flow immunological detection system for B. anthracis spores. This system involves the use of a portable magnetic assay reader, super-paramagnetic iron oxide particles, lateral-flow strips and two different monoclonal antibodies directed against B. anthracis spores. This detection system specifically recognises as few as 400 pure B. anthracis spores in 30 min. This system has a linear range of 4×10³-10⁶ CFU ml⁻¹ and reproducible detection limits of 200 spores mg⁻¹ milk powder and 130 spores mg⁻¹ soil for simulated samples. In addition, this approach shows no obvious cross-reaction with other related Bacillus spores, even at high concentrations, and has no significant dependence on the duration of the storage of the immunological strips. Therefore, this super-paramagnetic lateral-flow immunological detection system is a promising tool for the rapid and sensitive detection of Bacillus anthracis spores under field conditions. Copyright © 2012 Elsevier B.V. All rights reserved.

Seasonal variation of Ganoderma spore concentrations in urban and suburban districts of the city of Szczecin, Poland.

PubMed

Grinn-Gofroń, Agnieszka; Strzelczak, Agnieszka; Przestrzelska, Katarzyna

2015-01-01

According to recent studies, Ganoderma may be the third genus, after Alternaria and Cladosporium, the spores of which cause symptoms of allergy, and concentration is related to meteorological factors. The aerobiology of Ganoderma spores in Szczecin in urban and suburban districts was examined using Lanzoni Volumetric Spore Traps in 2008-2010. Ganoderma spores were present in the atmosphere on more than 90% of the days from June through September with peak concentrations in June, July and September. The number of days with spores was lower in the suburban district, while the total number of spores collected was higher there than in the urban district. Correlation and multiple regression analyses revealed weak relationships between Ganoderma and meteorological conditions, while testing the significance of differences between the districts showed that urban development did not have a clear impact on the values of meteorological parameters. A significantly higher abundance of spores in the suburbs of Szczecin seemed to be conditioned by the closeness of potential area sources. This study indicates that a single measuring site in the city centre insufficiently reflected the dynamics and level of Ganoderma spore concentration in peripheral districts.

The influence of lime and nitrogen fertilisers on spore counts of Pithomyces chartarum in pasture.

PubMed

Cuttance, E L; Laven, R A; Mason, W A; Stevenson, M

2016-11-01

To determine whether the application of lime or nitrogen to pasture affected the spore counts of Pithomyces chartarum. The lime application studies were undertaken on a spring-calving, pasture-based, commercial dairy farm near Te Awamutu, New Zealand. On 6 November 2012, five randomly selected paddocks were split into three equal sections. In two of the sections, lime was applied at either 1.5 or 2.5 t/ha, and the central section was left as an untreated control. Each section was sampled for spore counting weekly from 16 January to 15 May 2013. Starting in January 2013, five other randomly selected paddocks were monitored for spore counts. On 20 March 2013 the average spore counts in three paddocks were >100,000 spores/g of pasture. These paddocks were then divided into three equal sections and lime was applied as described above. Spore counting in each section continued weekly until 15 May 2013. The nitrogen application study was carried out on three commercial dairy farms near Te Awamutu, New Zealand. Two randomly selected paddocks on each farm were divided into three equal sections and, on 20 December 2012, nitrogen in the form of urea was applied at either 50 or 80 kg urea/ha to two of the sections; the central section remained as an untreated control. Each section was sampled for spore counting weekly from 16 January to 15 May 2013. Following pre-summer lime application, treatment at 1.5 or 2.5 t/ha did not affect spore counts over time compared with the control section (p>0.26). Similarly following autumn lime application, treatment at 1.5 or 2.5 t/ha did not affect spore counts over time compared with the control section (p>0.11). Following nitrogen application median spore counts remained <20,000 spores/g pasture throughout the trial period and there was no effect of treatment on spore counts over time (p>0.49). This study found that application of lime before the risk period for facial eczema, in November, application of lime after a spore count rise, in March, or urea application in December did not affect changes in number of spores produced by P. chartarum. This study does not support previous suggestions that fertilising pasture with lime or urea could alter the spore counts of P. chartarum. Fertiliser use does not provide an alternative to, or support, conventional methods of facial eczema control such as zinc prophylaxis or treatment of pasture with fungicides.

Stratosphere Conditions Inactivate Bacterial Endospores from a Mars Spacecraft Assembly Facility

PubMed Central

Khodadad, Christina L.; Wong, Gregory M.; James, Leandro M.; Thakrar, Prital J.; Lane, Michael A.; Catechis, John A.

2017-01-01

Abstract Every spacecraft sent to Mars is allowed to land viable microbial bioburden, including hardy endospore-forming bacteria resistant to environmental extremes. Earth's stratosphere is severely cold, dry, irradiated, and oligotrophic; it can be used as a stand-in location for predicting how stowaway microbes might respond to the martian surface. We launched E-MIST, a high-altitude NASA balloon payload on 10 October 2015 carrying known quantities of viable Bacillus pumilus SAFR-032 (4.07 × 107 spores per sample), a radiation-tolerant strain collected from a spacecraft assembly facility. The payload spent 8 h at ∼31 km above sea level, exposing bacterial spores to the stratosphere. We found that within 120 and 240 min, spore viability was significantly reduced by 2 and 4 orders of magnitude, respectively. By 480 min, <0.001% of spores carried to the stratosphere remained viable. Our balloon flight results predict that most terrestrial bacteria would be inactivated within the first sol on Mars if contaminated spacecraft surfaces receive direct sunlight. Unfortunately, an instrument malfunction prevented the acquisition of UV light measurements during our balloon mission. To make up for the absence of radiometer data, we calculated a stratosphere UV model and conducted ground tests with a 271.1 nm UVC light source (0.5 W/m2), observing a similarly rapid inactivation rate when using a lower number of contaminants (640 spores per sample). The starting concentration of spores and microconfiguration on hardware surfaces appeared to influence survivability outcomes in both experiments. With the relatively few spores that survived the stratosphere, we performed a resequencing analysis and identified three single nucleotide polymorphisms compared to unexposed controls. It is therefore plausible that bacteria enduring radiation-rich environments (e.g., Earth's upper atmosphere, interplanetary space, or the surface of Mars) may be pushed in evolutionarily consequential directions. Key Words: Planetary protection—Stratosphere—Balloon—Mars analog environment—E-MIST payload—Bacillus pumilus SAFR-032. Astrobiology 17, 337–350. PMID:28323456

Analysis of pulmonary surfactant by Fourier-transform infrared spectroscopy following exposure to Stachybotrys chartarum (atra) spores.

PubMed

McCrae, K C; Rand, T; Shaw, R A; Mason, C; Oulton, M R; Hastings, C; Cherlet, T; Thliveris, J A; Mantsch, H H; MacDonald, J; Scott, J E

2001-03-01

Lung cells are among the first tissues of the body to be exposed to air-borne environmental contaminants. Consequently the function of these cells may be altered before other cells are affected. As gas exchange takes place in the lungs, changes in cellular function may have serious implications for the processes of oxygen uptake and carbon dioxide elimination. In order for these processes to occur, the lung must maintain a high degree of expandability. This latter function is accomplished in part by the pulmonary surfactant which is synthesized and released by alveolar type II cells. Earlier studies have shown that exposure to gas phase materials such as smoke or organic solvents can alter the composition and function of the surfactant. The present study examines the ability of highly toxigenic mold spores to alter surfactant composition. Stachybotrys chartarum spores suspended in saline were instilled into mouse trachea as described earlier. After 24 h, the lungs were lavaged and the different processing stages of surfactant isolated by repeated centrifugation. Intracellular surfactant was isolated from the homogenized lung tissue by centrifugation on a discontinuous sucrose gradient. Samples were extracted into chloroform-methanol, dried and analyzed by Fourier-Transform infrared spectroscopy (FTIR). Exposure to S. chartarum induced an overall reduction of phospholipid among the three surfactant subfractions. The intermediate and spent surfactant fractions in particular were reduced to about half of the values observed in the saline-treated group. The relative distribution of phospholipid was also altered by spore exposure. Within the intracellular surfactant pool, higher levels of phospholipid were detected after spore exposure. In addition, changes were observed in the nature of the phospholipids. In particular strong intramolecular hydrogen bonding, together with other changes, suggested that spore exposure was associated with absence of an acyl chain esterified on the glycerol backbone, resulting in elevated levels of lysophospholipid in the samples. This study shows that mold spores and their products induce changes in regulation of both secretion and synthesis of surfactant, as well as alterations in the pattern of phospholipid targeting to the pulmonary surfactant pools.

Two distinct groups within the Bacillus subtilis group display significantly different spore heat resistance properties.

PubMed

Berendsen, Erwin M; Zwietering, Marcel H; Kuipers, Oscar P; Wells-Bennik, Marjon H J

2015-02-01

The survival of bacterial spores after heat treatment and the subsequent germination and outgrowth in a food product can lead to spoilage of the food product and economical losses. Prediction of time-temperature conditions that lead to sufficient inactivation requires access to detailed spore thermal inactivation kinetics of relevant model strains. In this study, the thermal inactivation kinetics of spores of fourteen strains belonging to the Bacillus subtilis group were determined in detail, using both batch heating in capillary tubes and continuous flow heating in a micro heater. The inactivation data were fitted using a log linear model. Based on the spore heat resistance data, two distinct groups (p < 0.001) within the B. subtilis group could be identified. One group of strains had spores with an average D120 °C of 0.33 s, while the spores of the other group displayed significantly higher heat resistances, with an average D120 °C of 45.7 s. When comparing spore inactivation data obtained using batch- and continuous flow heating, the z-values were significantly different, hence extrapolation from one system to the other was not justified. This study clearly shows that heat resistances of spores from different strains in the B. subtilis group can vary greatly. Strains can be separated into two groups, to which different spore heat inactivation kinetics apply. Copyright © 2014 Elsevier Ltd. All rights reserved.

Contribution of Spores to the Ability of Clostridium difficile To Adhere to Surfaces

PubMed Central

Joshi, Lovleen Tina; Phillips, Daniel S.; Williams, Catrin F.; Alyousef, Abdullah

2012-01-01

Clostridium difficile is the commonest cause of hospital-acquired infection in the United Kingdom. We characterized the abilities of 21 clinical isolates to form spores; to adhere to inorganic and organic surfaces, including stainless steel and human adenocarcinoma cells; and to germinate. The composition of culture media had a significant effect on spore formation, as significantly more spores were produced in brain heart infusion broth (Student's t test; P = 0.018). The spore surface relative hydrophobicity (RH) varied markedly (14 to 77%) and was correlated with the ability to adhere to stainless steel. We observed no correlation between the ribotype and the ability to adhere to steel. When the binding of hydrophobic (DS1813; ribotype 027; RH, 77%) and hydrophilic (DS1748; ribotype 002; RH, 14%) spores to human gut epithelial cells at different stages of cell development was examined, DS1813 spores adhered more strongly, suggesting the presence of surface properties that aid attachment to human cells. Electron microscopy studies revealed the presence of an exosporium surrounding DS1813 spores that was absent from spores of DS1748. Finally, the ability of spores to germinate was found to be strain and medium dependent. While the significance of these findings to the disease process has yet to be determined, this study has highlighted the importance of analyzing multiple isolates when attempting to characterize the behavior of a bacterial species. PMID:22923404

Comparison of Fe(VI) (FeO4(2-)) and ozone in inactivating Bacillus subtilis spores.

PubMed

Makky, Essam A; Park, Gui-Su; Choi, Ik-Won; Cho, Sung-Il; Kim, Hyunook

2011-05-01

The protozoan parasites such as Cryptosporidiumparvum and Giardialamblia have been recognized as a frequent cause of recent waterborne disease outbreaks because of their strong resistance against chlorine disinfection. In this study, ozone and Fe(VI) (i.e., FeO(4)(2-)) were compared in terms of inactivation efficiency for Bacillus subtilis spores which are commonly utilized as an indicator of protozoan pathogens. Both oxidants highly depended on water pH and temperature in the spore inactivation. Since redox potential of Fe(VI) is almost the same as that of ozone, spore inactivation efficiency of Fe(VI) was expected to be similar with that of ozone. However, it was found that ozone was definitely superior over Fe(VI): at pH 7 and 20°C, ozone with the product of concentration×contact time (C¯T) of 10mgL(-1)min inactivate the spores more than 99.9% within 10min, while Fe(VI) with C¯T of 30mgL(-1) min could inactivate 90% spores. The large difference between ozone and Fe(VI) in spore inactivation was attributed mainly to Fe(III) produced from Fe(VI) decomposition at the spore coat layer which might coagulate spores and make it difficult for free Fe(VI) to attack live spores. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

A Novel Protocol for Decoating and Permeabilizing Bacterial Spores for Epifluorescent Microscopy

NASA Technical Reports Server (NTRS)

LaDuc, Myron T.; Mohapatra, Bidyut

2014-01-01

Based on previously reported procedures for permeabilizing vegetative bacterial cells, and numerous trial-and-error attempts with bacterial endospores, a protocol was developed for effectively permeabilizing bacterial spores, which facilitated the applicability of fluorescent in situ hybridization (FISH) microscopy. Bacterial endospores were first purified from overgrown, sporulated suspensions of B. pumilus SAFR-032. Purified spores at a concentration of approx equals 10 million spores/mL then underwent proteinase-K treatment, in a solution of 468.5 µL of 100 mM Tris-HCl, 30 µL of 10% SDS, and 1.5 microL of 20 mg/mL proteinase-K for ten minutes at 35 ºC. Spores were then harvested by centrifugation (15,000 g for 15 minutes) and washed twice with sterile phosphate-buffered saline (PBS) solution. This washing process consisted of resuspending the spore pellets in 0.5 mL of PBS, vortexing momentarily, and harvesting again by centrifugation. Treated and washed spore pellets were then resuspended in 0.5 mL of decoating solution, which consisted of 4.8 g urea, 3 mL Milli-Q water, 1 mL 0.5M Tris, 1 mL 1M dithiothreitol (DTT), and 2 mL 10% sodium-dodecylsulfate (SDS), and were incubated at 65 ºC for 15 minutes while being shaken at 165 rpm. Decoated spores were then, once again, washed twice with sterile PBS, and subjected to lysozyme/mutanolysin treatment (7 mg/mL lysozyme and 7U mutanolysin) for 15 minutes at 35 C. Spores were again washed twice with sterile PBS, and spore pellets were resuspended in 1-mL of 2% SDS. This treatment, facilitating inner membrane permeabilization, lasted for ten minutes at room temperature. Permeabilized spores were washed two final times with PBS, and were resuspended in 200 mkcroL of sterile PBS. At this point, the spores were permeable and ready for downstream processing, such as oligonucleotideprobe infiltration, hybridization, and microscopic evaluation. FISH-microscopic imagery confirmed the effective and efficient (˜50% successful permeabilization and recovery) permeabilization of numerous spore preparations. The novelty of the technology developed here is in its applicability to bacterial endospores. While protocols abound for the effective permeabilization of bacterial, archaeal, and eukaryotic vegetative cells, there are no such reliable methods for decoating and permeabilizing bacterial endospores in a manner that is amenable to downstream FISH microscopic analyses. This innovation enables the direct visualization and enumeration of spores via FISH-based microscopic techniques, circumventing the complications that accompany previously required germination regimes. The synergistic enzymatic weakening of the many spore layers facilitates a structural compromise that is just enough to render the spores permeable without degrading the spore to a level, which precludes it from recognition.

Fungal microcolonies on indoor surfaces — an explanation for the base-level fungal spore counts in indoor air

NASA Astrophysics Data System (ADS)

Pasanen, A.-L.; Heinonen-Tanski, H.; Kalliokoski, P.; Jantunen, M. J.

In the subarctic winter, fungal spores are found in indoor air even when outdoor spore levels are very low. The results of this study support an explanation that some indoor airborne fungal spores are derived from unnoticeable fungal microcolonies, which may develop on temporarily wet surfaces. Laboratory experiments on Penicillium verrucosum indicated that the fungus germinated on new wallpaper very quickly (about half an hour) under moist conditions. Hyphal growth and sporulation of the fungus on moist wallpaper occured within one day of incubation. In gravity-settling tape samples from occasionally wet surfaces in a suburban home, large spore aggregates, hyphal fragments with some spores and spores in the germination stage were found, indicating fungal growth. These experiments showed that fungal microcolonies can develop within a week on occasionally wet indoor surfaces.

Bacillus anthracis spores germinate extracellularly at air-liquid interface in an in vitro lung model under serum-free conditions

DOE PAGES

Powell, Joshua D.; Hutchison, Janine R.; Hess, Becky M.; ...

2015-07-30

Aims: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. Methods and Results: We evaluated the kinetics of uptake, germination and proliferation of B. anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3, and A549 cell lines. We also analyzed the influence of various cell culture media formulations related to spore germination. Conclusions: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the extracellular environment was evident, and was appreciably higher in A549 and Calu-3 cultures than in primary epithelial cells. Additionally, ourmore » results revealed spores in association with primary cells submerged in cell culture media germinated 1 h« less

Germination of Spores of Astrobiologically Relevant Bacillus Species in High-Salinity Environments

NASA Astrophysics Data System (ADS)

Nagler, Katja; Julius, Christina; Moeller, Ralf

2016-07-01

In times of increasing space exploration and search for extraterrestrial life, new questions and challenges for planetary protection, aiming to avoid forward contamination of different planets or moons with terrestrial life, are emerging. Spore-forming bacteria such as Bacillus species have a high contamination potential due to their spores' extreme resistance, enabling them to withstand space conditions. Spores require liquid water for their conversion into a growing cell (i.e., spore germination and subsequent growth). If present, water on extraterrestrial planets or moons is likely to be closely associated with salts (e.g., in salty oceans or brines), thus constituting high-salinity environments. Spores of Bacillus subtilis can germinate despite very high salt concentrations, although salt stress does exert negative effects on this process. In this study, germination and metabolic reactivation ("outgrowth") of spores of five astrobiologically relevant Bacillus species (B. megaterium, B. pumilus SAFR-032, B. nealsonii, B. mojavensis, and B. vallismortis) in high salinity (≤3.6 M NaCl) were investigated. Spores of different species exhibited different germination and outgrowth capabilities in high salinity, which strongly depended on germination conditions, especially the exact composition of the medium. In this context, a new "universal" germination trigger for Bacillus spores, named KAGE (KCl, L-alanine, D-glucose, ectoine), was identified, which will be very useful for future comparative germination and outgrowth studies on different Bacillus species. Overall, this study yielded interesting new insights on salt stress effects on spore germination and points out the difficulty of predicting the potential of spores to contaminate salty environments on extraterrestrial celestial bodies.

Decontamination of fluid milk containing Bacillus spores using commercial household products.

PubMed

Black, D G; Taylor, T M; Kerr, H J; Padhi, S; Montville, T J; Davidson, P M

2008-03-01

Although commercial sanitizers can inactivate bacterial spores in food processing environments, relatively little data exist as to the decontamination of products and surfaces by consumers using commercial household products. Should a large scale bioterrorism incident occur in which consumer food products were contaminated with a pathogenic sporeformer such as Bacillus anthracis, there may be a need to decontaminate these products before disposal as liquid or solid waste. Studies were conducted to test the efficacy of commercial household products for inactivating spores of Bacillus cereus (used as a surrogate for B. anthracis) in vitro and in fluid milk. Validation of the resistance of the B. cereus spores was confirmed with B. anthracis spores. Fifteen commercial products, designed as either disinfectants or sanitizers or as potential sanitizers, were purchased from retail markets. Products selected had one of the following active compounds: NaOCl, HCl, H2O2, acetic acid, quaternary ammonium compounds, ammonium hydroxide, citric acid, isopropanol, NaOH, or pine oil. Compounds were diluted in water (in vitro) or in 2% fat fluid milk, and spores were exposed for up to 6 h. Products containing hypochlorite were most effective against B. cereus spores. Products containing HCl or H2O2 also reduced significant numbers of spores but at a slower rate. The resistance of spores of surrogate B. cereus strains to chlorine-containing compounds was similar to that of B. anthracis spores. Therefore, several household products on the market may be used to decontaminate fluid milk or similar food products contaminated by spores of B. anthracis.

FACTORS RELATING TO THE RELEASE OF STACHYBOTRYS CHARTARUM SPORES FROM CONTAMINATED SOURCES

EPA Science Inventory

The paper describes preliminary results of a research project to determine the factors that control the release of S. chartarum spores from a contaminated source and test ways to reduce spore release and thus exposure. As anticipated, S. chartarum spore emissions from gypsum boar...

9 CFR 113.66 - Anthrax Spore Vaccine-Nonencapsulated.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Anthrax Spore Vaccine-Nonencapsulated... REQUIREMENTS Live Bacterial Vaccines § 113.66 Anthrax Spore Vaccine—Nonencapsulated. Anthrax Spore Vaccine.... All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed...

9 CFR 113.66 - Anthrax Spore Vaccine-Nonencapsulated.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Anthrax Spore Vaccine-Nonencapsulated... REQUIREMENTS Live Bacterial Vaccines § 113.66 Anthrax Spore Vaccine—Nonencapsulated. Anthrax Spore Vaccine.... All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed...

9 CFR 113.66 - Anthrax Spore Vaccine-Nonencapsulated.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Anthrax Spore Vaccine-Nonencapsulated... REQUIREMENTS Live Bacterial Vaccines § 113.66 Anthrax Spore Vaccine—Nonencapsulated. Anthrax Spore Vaccine.... All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed...

9 CFR 113.66 - Anthrax Spore Vaccine-Nonencapsulated.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Anthrax Spore Vaccine-Nonencapsulated... REQUIREMENTS Live Bacterial Vaccines § 113.66 Anthrax Spore Vaccine—Nonencapsulated. Anthrax Spore Vaccine.... All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed...

Effects of nifedipine on gravi-dependent germination of moss spores

NASA Astrophysics Data System (ADS)

Khorkavtsiv, O. Y.; Demkiv, O. T.

Influence of gravity on germination of spores and dependence of the generation of a polar axis on a Ca2+ influx were investigated. The germination of spores does not depend on gravity but outgrowth polarity is controlled by light and gravity (Sytnik et al., 1989; Pundiak et al., 2001). We have shown that gravity determines the polarity of germination of spores and development of rhizoid and chloronemal outgrowths in both moss species -- Ceratodon purpureus and Pohlia nutans, the alignment of polar of germinating spores in C. purpureus, however, is less dependent on gravistimulus than in P. nutans. In 48 h after sowing onto culture medium+0,2% glucose in vertically oriented petri dishes in darkness spores of P. nutans germinated positively gravitropic rhizoid at the lower spore side and negatively gravitropic chloronema at the opposite one. The germination of C. purpureus spores is similar but the outgrowths show the lower level of alignment to the gravity vector than that of P. nutans, the dispersion of angles being 8,9 vs. 1,2 respectively. The cellular mechanism by which gravity acts remains unknown. The intracellular signaling Ca2+ ions play a crucial role in gravity perception and ability of a single cell to respond to gravity. We determined relative intensity of Ca2+ luminescence in the spores before their germination and at the early stages of outgrowth formation after treatment with the nifedipine and in a dependence on gravity vector. Gravity determined the position of outgrowth initiation zone and later on the growth direction of spore filaments. Treatment with nifedipine suppressed the gravity-directed calcium channel influx and distrupted polar growth of outgrowths. In experiments with calcium channel blocker sterilized spores were pregerminated on normal Knop's agar one day after were transferred to 50 μ M nifedipine just before emergence of the germ tube. After 48 h on nifedipine treatment, 50% spores did not germinate, 35% grew apolarily and in 15% of spores cell filaments oriented parallely with respect to the gravity vector. Results shown suggest that the endogenic competency of a single-cell spore is necessary condition of gravi- induced initiation of polar axis the competency being realized with Ca2+ movement. The highest level of Ca2+ luminescence was at the bottom of spores. In other sites of the spores the Ca2+ luminescence was about 20-fold lower than at the site of Ca2+ influx. In the 24 h after formation of first outgrowth the new site of Ca2+ influx appeared at the opposite site of spore and the second outgrowth arised. Consequently during the period of gravi-dependent spore development the newly top Ca2+ influx was repeatedly established. The direction of the Ca2+ ions influx correlated with re-orientation of spores with respect to the gravity vector. It is known that the nifedipine partially inhibits polar axis formation (Chatterjee et al., 2000) the latter being formed under the influence Ca2+ gradient (Cove, 2000). Thus, our results confirm that the fast change of Ca2+ influx probably is one of the earliest cell-level responses induced by gravity and it plays a key role in guiding polar events of germinating spores. This research was supported by NASA grant NN-09 (R).

Detection of chaos: New approach to atmospheric pollen time-series analysis

NASA Astrophysics Data System (ADS)

Bianchi, M. M.; Arizmendi, C. M.; Sanchez, J. R.

1992-09-01

Pollen and spores are biological particles that are ubiquitous to the atmosphere and are pathologically significant, causing plant diseases and inhalant allergies. One of the main objectives of aerobiological surveys is forecasting. Prediction models are required in order to apply aerobiological knowledge to medical or agricultural practice; a necessary condition of these models is not to be chaotic. The existence of chaos is detected through the analysis of a time series. The time series comprises hourly counts of atmospheric pollen grains obtained using a Burkard spore trap from 1987 to 1989 at Mar del Plata. Abraham's method to obtain the correlation dimension was applied. A low and fractal dimension shows chaotic dynamics. The predictability of models for atomspheric pollen forecasting is discussed.

Ultrastructural and molecular characterization of Glugea serranus n. sp., a microsporidian infecting the blacktail comber, Serranus atricauda (Teleostei: Serranidae), in the Madeira Archipelago (Portugal).

PubMed

Casal, Graça; Rocha, Sónia; Costa, Graça; Al-Quraishy, Saleh; Azevedo, Carlos

2016-10-01

A new microsporidian infecting the connective tissue of the coelomic cavity of the blacktail comber Serranus atricauda, in the Madeira Archipelago (Portugal), is described on the basis of morphological, ultrastructural, and molecular features. The microsporidian formed large whitish xenomas adhering to the peritoneal visceral organs of the host. Each xenoma consisted of a single hypertrophic cell, in the cytoplasm of which mature spores proliferated within parasitophorous vacuoles surrounded by numerous collagen fibers. Mature spores were ellipsoidal and uninucleated, measuring an average of 6.5 ± 0.5 μm in length and 3.4 ± 0.6 μm in width. The anchoring disk of the polar filament was subterminal, laterally shifted from the anterior pole of the spore. The isofilar polar filament coiled in 18-19 turns, forming two rows that surrounded the posterior vacuole. The latter occupied about one third of the spore length. The polaroplast surrounding the apical and uncoiled portion of the polar filament displayed two distinct regions: a lamellar region and an electron-dense globule. Molecular analysis of the rRNA genes, including the internal transcribed spacer region, and phylogenetic analysis using maximum likelihood and neighbor joining demonstrated that this microsporidian parasite clustered with some Glugea species. Based on the differences found both at the morphological and molecular levels, to other members of the genus Glugea, the microsporidian infecting the blacktail comber is considered a new species, thus named Glugea serranus n. sp.

Determining the Effects of High Intensity Ultrasound on the Reduction of Microbes in Milk and Orange Juice Using Response Surface Methodology.

PubMed

Ganesan, Balasubramanian; Martini, Silvana; Solorio, Jonathan; Walsh, Marie K

2015-01-01

This study investigated the effects of high intensity ultrasound (temperature, amplitude, and time) on the inactivation of indigenous bacteria in pasteurized milk, Bacillus atrophaeus spores inoculated into sterile milk, and Saccharomyces cerevisiae inoculated into sterile orange juice using response surface methodology. The variables investigated were sonication temperature (range from 0 to 84°C), amplitude (range from 0 to 216 μm), and time (range from 0.17 to 5 min) on the response, log microbe reduction. Data were analyzed by statistical analysis system software and three models were developed, each for bacteria, spore, and yeast reduction. Regression analysis identified sonication temperature and amplitude to be significant variables on microbe reduction. Optimization of the inactivation of microbes was found to be at 84.8°C, 216 μm amplitude, and 5.8 min. In addition, the predicted log reductions of microbes at common processing conditions (72°C for 20 sec) using 216 μm amplitude were computed. The experimental responses for bacteria, spore, and yeast reductions fell within the predicted levels, confirming the accuracy of the models.

Bacillus nealsonii sp. nov., isolated from a spacecraft-assembly facility, whose spores are gamma-radiation resistant

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri; Kempf, Michael; Chen, Fei; Satomi, Masataka; Nicholson, Wayne; Kern, Roger

2003-01-01

One of the spore-formers isolated from a spacecraft-assembly facility, belonging to the genus Bacillus, is described on the basis of phenotypic characterization, 16S rDNA sequence analysis and DNA-DNA hybridization studies. It is a Gram-positive, facultatively anaerobic, rod-shaped eubacterium that produces endospores. The spores of this novel bacterial species exhibited resistance to UV, gamma-radiation, H2O2 and desiccation. The 18S rDNA sequence analysis revealed a clear affiliation between this strain and members of the low G+C Firmicutes. High 16S rDNA sequence similarity values were found with members of the genus Bacillus and this was supported by fatty acid profiles. The 16S rDNA sequence similarity between strain FO-92T and Bacillus benzoevorans DSM 5391T was very high. However, molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in this genus, but DNA-DNA hybridization data support the proposal of FO-92T as Bacillus nealsonii sp. nov. (type strain is FO-92T =ATCC BAAM-519T =DSM 15077T).

Determining the Effects of High Intensity Ultrasound on the Reduction of Microbes in Milk and Orange Juice Using Response Surface Methodology

PubMed Central

Martini, Silvana; Solorio, Jonathan; Walsh, Marie K.

2015-01-01

This study investigated the effects of high intensity ultrasound (temperature, amplitude, and time) on the inactivation of indigenous bacteria in pasteurized milk, Bacillus atrophaeus spores inoculated into sterile milk, and Saccharomyces cerevisiae inoculated into sterile orange juice using response surface methodology. The variables investigated were sonication temperature (range from 0 to 84°C), amplitude (range from 0 to 216 μm), and time (range from 0.17 to 5 min) on the response, log microbe reduction. Data were analyzed by statistical analysis system software and three models were developed, each for bacteria, spore, and yeast reduction. Regression analysis identified sonication temperature and amplitude to be significant variables on microbe reduction. Optimization of the inactivation of microbes was found to be at 84.8°C, 216 μm amplitude, and 5.8 min. In addition, the predicted log reductions of microbes at common processing conditions (72°C for 20 sec) using 216 μm amplitude were computed. The experimental responses for bacteria, spore, and yeast reductions fell within the predicted levels, confirming the accuracy of the models. PMID:26904659

Electrical Sensing Zone Particle Analyzer for Measuring Germination of Fungal Spores in the Presence of Other Particles1

PubMed Central

Santoro, T.; Stotzky, G.; Rem, L. T.

1967-01-01

Microscopic, respirometric, and electronic sizing methods for measuring germination of fungal spores were compared. With the electronic sizing method, early stages of germination (i.e., spore swelling) were detected long before germ tube emergence or significant changes in respiratory rates were observed. This method, which is rapid, easy, sensitive, and reproducible, also permits measuring the germination of spores when similar-size particles are present in concentrations considerably in excess of the number of spores. PMID:6069161

DOE Office of Scientific and Technical Information (OSTI.GOV)

Powell, Joshua D.; Hutchison, Janine R.; Hess, Becky M.

Aims: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. Methods and Results: We evaluated the kinetics of uptake, germination and proliferation of B. anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3, and A549 cell lines. We also analyzed the influence of various cell culture media formulations related to spore germination. Conclusions: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the extracellular environment was evident, and was appreciably higher in A549 and Calu-3 cultures than in primary epithelial cells. Additionally, ourmore » results revealed spores in association with primary cells submerged in cell culture media germinated 1 h« less

Survival strategies of Bacillus spores in food.

PubMed

Stecchini, Mara Lucia; Del Torre, Manuela; Polese, Pierluigi

2013-11-01

Control of bacterial spores is one of the major problem in the food preservation. Spores of Bacillus genus are commonly present in different environments, including soil and the gut of insects and animals and, as a result, they can be spread to all kind of foods. Due to their high resistance properties, their complete inactivation in food is often impossible without changing the product characteristics. Surviving spores can germinate and grow out to vegetative cells, with the consequent great risk of food spoilage and food poisoning after consumption. Spores have evolved various mechanisms, including phenotypic variability, to protect themselves from a wide range of damage resulting from food preservation treatments. Even if the phenotypic heterogeneity contributes to increase the chances of survival of Bacillus spore to conventional preservation treatments, in some specific instances, an homogeneous response could be the result of a strategy adopted by the spores to increase resistance to those treatments.

Microbial Burden Approach : New Monitoring Approach for Measuring Microbial Burden

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri; Vaishampayan, Parag; Barmatz, Martin

2013-01-01

Advantages of new approach for differentiating live cells/ spores from dead cells/spores. Four examples of Salmonella outbreaks leading to costly destruction of dairy products. List of possible collaboration activities between JPL and other industries (for future discussion). Limitations of traditional microbial monitoring approaches. Introduction to new approach for rapid measurement of viable (live) bacterial cells/spores and its areas of application. Detailed example for determining live spores using new approach (similar procedure for determining live cells). JPL has developed a patented approach for measuring amount of live and dead cells/spores. This novel "molecular" method takes less than 5 to 7 hrs. compared to the seven days required using conventional techniques. Conventional "molecular" techniques can not discriminate live cells/spores among dead cells/spores. The JPL-developed novel method eliminates false positive results obtained from conventional "molecular" techniques that lead to unnecessary delay in the processing and to unnecessary destruction of food products.

Pharmacological Properties of Biocompounds from Spores of the Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes): A Review.

PubMed

Soccol, Carlos Ricardo; Bissoqui, Lucas Yamasaki; Rodrigues, Cristine; Rubel, Rosalia; Sella, Sandra R B R; Leifa, Fan; de Souza Vandenberghe, Luciana Porto; Soccol, Vanete Thomaz

2016-01-01

Ganoderma lucidum is a well-known representative of mushrooms that have been used in traditional Chinese medicine for centuries. New discoveries related to this medicinal mushroom and its biological properties are frequently reported. However, only recently have scientists started to pay special attention to G. lucidum spores. This is in part because of the recent development of methods for breaking the spore wall and extracting biocompounds from the spore. Although some research groups are working with G. lucidum spores, data in the literature are still limited, and the methods used have not been systematized. This review therefore describes the main advances in techniques for breaking the spore wall and extracting biocompounds from the spore. In addition, the major active components identified and their biological properties, such as neurological activity and antiaging and cell-protective effects, are investigated because these are of importance for potential drug development.

Differential survival of Ichthyophonus isolates indicates parasite adaptation to its host environment

USGS Publications Warehouse

Hershberger, P.K.; Pacheco, C.A.; Gregg, J.L.; Purcell, M.K.; LaPatra, S.E.

2008-01-01

In vitro viability of Ichthyophonus spp. spores in seawater and freshwater corresponded with the water type of the host from which the spores were isolated. Among Ichthyophonus spp. spores from both marine and freshwater fish hosts (Pacific herring, Clupea pallasii, and rainbow trout, Oncorhynchus mykiss, respectively), viability was significantly greater (P < 0.05) after incubation in seawater than in freshwater at all time points from 1 to 60 min after immersion; however, magnitude of the spore tolerances to water type differed with host origin. Ichthyophonus sp. adaptation to its host environment was indicated by greater seawater tolerance of spores from the marine host and greater freshwater tolerance of spores from the freshwater host. Prolonged aqueous survival of Ichthyophonus spp. spores in the absence of a host provides insight into routes of transmission, particularly among planktivorous fishes, and should be considered when designing strategies to dispose of infected fish carcasses and tissues.

Cryopreservation of spores of Dicksonia sellowiana: an endangered tree fern indigenous to South and Central America.

PubMed

Rogge, G D; Viana, A M; Randi, A M

2000-01-01

Spores of Dicksonia sellowiana (Presl.) Hook., an endangered tree fern, were stored in liquid nitrogen. Surface sterilized spores were placed in 1 ml sterile polypropylene cryotubes and were plunged into liquid nitrogen cryo-cans for 15 minutes, 15 days, 1 month and 3 months. In all, of the treatments the percentage of germination was higher than the control (fresh spores). Germination in Dyer and MS media supplement with 10 (-7) M and 5 x 10(-7) M BA was also promoted as comparing to control. There was no difference between the germination of spores thawed rapidly in a water bath at 45 degree C during 5 minutes or slowly at room temperature. Cryopreservation seems to promote germination of some dormant spores of D. sellowiana. The pre-treatment in cryoprotective solution of dimethyl sulphoxide 15%(v/v) in 1 M glycerol inhibited the germination of cryopreserved spores

Recovery of Phakopsora pachyrhizi urediniospores from Passive Spore Trap Slides and Extraction of Their DNA for Quantitative PCR

USDA-ARS?s Scientific Manuscript database

Enumeration of rust spores from passive spore traps utilizing white petrolatum-coated slides by traditional microscopic evaluation can represent a serious challenge. Many fungal spores look alike, and clear visualization on the adhesive can be obscured by particulate debris or nonuniformities within...

Anthrax Spores under a microscope

NASA Technical Reports Server (NTRS)

2003-01-01

Anthrax spores are inactive forms of Bacillus anthracis. They can survive for decades inside a spore's tough protective coating; they become active when inhaled by humans. A result of NASA- and industry-sponsored research to develop small greenhouses for space research is the unique AiroCide TiO2 system that kills anthrax spores and other pathogens.

Occurrence of Cladosporium spp. and Alternaria spp. spores in Western, Northern and Central-Eastern Poland in 2004-2006 and relation to some meteorological factors

NASA Astrophysics Data System (ADS)

Grinn-Gofroń, Agnieszka; Rapiejko, Piotr

2009-08-01

The concentration of airborne spores of Cladosporium spp. and Alternaria spp. has been investigated at three monitoring stations situated along the west-north and central-east transect in Poland (Szczecin, Olsztyn, Warszawa,) i.e. from a height of 100 m to 149 m above sea level. The aerobiological monitoring of fungal spores was performed by means of three Lanzoni volumetric spore traps. Cladosporium spp. spores were dominant at all the stations. The highest Cladosporium spp. and Alternaria spp. numbers of spores were observed at all the cities in July and August. Statistically significant correlations have been found between the Cladosporium spp. and Alternaria spp. concentration in the air and the mean air temperature, amount of precipitation, air pressure and relative air humidity. The spore count of Cladosporium spp. and Alternaria spp. is determined by the diversity of local flora and weather conditions, especially by the air temperature. The identification of factors, which influence and shape spore concentrations, may significantly improve the current methods of allergy prevention.

Formation of Protoplasts from Resting Spores

PubMed Central

Fitz-James, Philip C.

1971-01-01

Coat-stripped spores suspended in hypertonic solutions and supplied with two essential cations can be converted into viable protoplasts by lysozyme digestion of both cortex and germ cell wall. Calcium ions are necessary to prevent membrane rupture, and magnesium ions are necessary for changes indicative of hydration of the core, particularily the nuclear mass. Since remnant spore coat covered such protoplasts of Bacillus subtilis and the germ cell wall of B. cereus spores is not lysozyme digestible, coatless spores of B. megaterium KM were more useful for these studies. Lysozyme digestion in cation-free environment produced a peculiar semi-refractile spore core free of a cortex but prone to rapid hydration and lytic changes on the addition of cations. Strontium could replace Ca2+ but Mn2+ could not replace Mg2+ in these digestions. When added to the spores, dipicolinic acid and other chelates appeared to compete with the membrane for the calcium needed for stabilization during lysozyme conversion to protoplasts. It is argued that calcium could function to stabilize the inner membrane anionic groups over the anhydrous dipicolinic acid-containing core of resting spores. Images PMID:4995380

Deposition of Bacillus subtilis spores using an airbrush-spray or spots to study surface decontamination by pulsed light.

PubMed

Levy, Caroline; Bornard, Isabelle; Carlin, Frédéric

2011-02-01

Microbial contamination on surfaces of food processing equipment is a major concern in industries. A new method to inoculate a single-cell layer (monolayer) of microorganisms onto polystyrene was developed, using a deposition with an airbrush. A homogeneous dispersion of Bacillus subtilis DSM 402 spores sprayed on the surface was observed using both plate count and scanning electron microscopy. No clusters were found, even with high spore concentrations (10(7) spores/inoculated surface). A monolayer of microorganisms was also obtained after deposition of 10 μL droplets containing 3×10(4) spores/spot on polystyrene disks, but not with a higher spore concentration. Pulsed light (PL) applied to monolayers of B. subtilis spores allowed log reductions higher than 6. As a consequence of clusters formation in spots of 10 μL containing more than 3×10(5) spores, log reductions obtained by PL were significantly lower. The comparative advantages of spot and spray depositions were discussed. Copyright © 2010 Elsevier B.V. All rights reserved.

Caenorhabditis elegans Predation on Bacillus anthracis: Decontamination of Spore Contaminated Soil with Germinants and Nematodes.

PubMed

Schelkle, Bettina; Choi, Young; Baillie, Leslie W; Richter, William; Buyuk, Fatih; Celik, Elif; Wendling, Morgan; Sahin, Mitat; Gallagher, Theresa

2017-01-01

Remediation of Bacillus anthracis -contaminated soil is challenging and approaches to reduce overall spore levels in environmentally contaminated soil or after intentional release of the infectious disease agent in a safe, low-cost manner are needed. B. anthracis spores are highly resistant to biocides, but once germinated they become susceptible to traditional biocides or potentially even natural predators such as nematodes in the soil environment. Here, we describe a two-step approach to reducing B. anthracis spore load in soil during laboratory trials, whereby germinants and Caenorhabditis elegans nematodes are applied concurrently. While the application of germinants reduced B. anthracis spore load by up to four logs depending on soil type, the addition of nematodes achieved a further log reduction in spore count. These laboratory based results suggest that the combined use of nematodes and germinants could represent a promising approach for the remediation of B. anthracis spore contaminated soil. Originality-Significance Statement: This study demonstrates for the first time the successful use of environmentally friendly decontamination methods to inactivate Bacillus anthracis spores in soil using natural predators of the bacterium, nematode worms.

Assessment of bacterial endospore viability with fluorescent dyes.

PubMed

Laflamme, C; Lavigne, S; Ho, J; Duchaine, C

2004-01-01

To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores. Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B. coagulans and two strains of B. subtilis) were used. The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC4(3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively. Gamma irradiation treatment completely eliminated spore culturability and was used as negative control. The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h. The membrane potential assessment gave no answer about spore viability. A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P < 0.02). It is possible to use CTC and Live/Dead BacLight to rapidly test endospore viability and evaluate the proportion of spores in a preparation that could not be recovered with plate count. This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.

Effects of superheated steam on Geobacillus stearothermophilus spore viability.

PubMed

Head, D S; Cenkowski, S; Holley, R; Blank, G

2008-04-01

To examine the effect of processing with superheated steam (SS) on Geobacillus stearothermophilus ATCC 10149 spores. Two inoculum levels of spores of G. stearothermophilus were mixed with sterile sand and exposed to SS at 105-175 degrees C. The decimal reduction time (D-value) and the thermal resistance constant (z-value) were calculated. The effect of cooling of spores between periods of exposure to SS was also examined. A mean z-value of 25.4 degrees C was calculated for both inoculum levels for SS processing temperatures between 130 degrees C and 175 degrees C. Spore response to SS treatment depends on inoculum size. SS treatment may be effective for reduction in viability of thermally resistant bacterial spores provided treatments are separated by intermittent cooling periods. There is a need for technologies that require short thermal processing times to eliminate bacterial spores in foods. The SS processing technique has the potential to reduce microbial load and to modify food texture with less energy in comparison to commonly used hot air treatment. This work provides information on the effect of SS processing parameters on the viability of G. stearothermophilus spores.

Evaluating novel synthetic compounds active against Bacillus subtilis and Bacillus cereus spores using Live imaging with SporeTrackerX.

PubMed

Omardien, Soraya; Ter Beek, Alexander; Vischer, Norbert; Montijn, Roy; Schuren, Frank; Brul, Stanley

2018-06-14

An empirical approach was taken to screen a novel synthetic compound library designed to be active against Gram-positive bacteria. We obtained five compounds that were active against spores from the model organism Bacillus subtilis and the food-borne pathogen Bacillus cereus during our population based experiments. Using single cell live imaging we were able to observe effects of the compounds on spore germination and outgrowth. Difference in sensitivity to the compounds could be observed between B. subtilis and B. cereus using live imaging, with minor difference in the minimal inhibitory and bactericidal concentrations of the compounds against the spores. The compounds all delayed the bursting time of germinated spores and affected the generation time of vegetative cells at sub-inhibitory concentrations. At inhibitory concentrations spore outgrowth was prevented. One compound showed an unexpected potential for preventing spore germination at inhibitory concentrations, which merits further investigation. Our study shows the valuable role single cell live imaging can play in the final selection process of antimicrobial compounds.

Hydrazine vapor inactivates Bacillus spores

NASA Astrophysics Data System (ADS)

Schubert, Wayne W.; Engler, Diane L.; Beaudet, Robert A.

2016-05-01

NASA policy restricts the total number of bacterial spores that can remain on a spacecraft traveling to any planetary body which might harbor life or have evidence of past life. Hydrazine, N2H4, is commonly used as a propellant on spacecraft. Hydrazine as a liquid is known to inactivate bacterial spores. We have now verified that hydrazine vapor also inactivates bacterial spores. After Bacillus atrophaeus ATCC 9372 spores deposited on stainless steel coupons were exposed to saturated hydrazine vapor in closed containers, the spores were recovered from the coupons, serially diluted, pour plated and the surviving bacterial colonies were counted. The exposure times required to reduce the spore population by a factor of ten, known as the D-value, were 4.70 ± 0.50 h at 25 °C and 2.85 ± 0.13 h at 35 °C. These inactivation rates are short enough to ensure that the bioburden of the surfaces and volumes would be negligible after prolonged exposure to hydrazine vapor. Thus, all the propellant tubing and internal tank surfaces exposed to hydrazine vapor do not contribute to the total spore count.

Cryogenic Irradiation of Bacillus Atrophaeus spores to understand microbial survival on Icy Bodies

NASA Astrophysics Data System (ADS)

Yerby, C. J.; Noell, A. C.; Hodyss, R. P.; Johnson, P. V.; Ponce, A.

2017-12-01

Bacterial Spores are useful indicator organisms for studying the survival of microbes and degradation of biomolecules on the surface of planetary icy bodies. To predict the limits of life's proliferation in space, specifically on icy bodies, it is essential to understand the ability of microbes to withstand photon and particle irradiation at cryogenic temperatures. Bacillus Atrophaeus spores were transferred onto stainless steel coupons by varied processes and subsequently frozen at Europan temperatures (16oK—273oK) in a vacuum at 8.7x10-8 Torr. An argon lamp bombarded the spore-containing coupons with a solar-like radiation spectra for a variety of times, and spores were removed from the coupons and enumerated in culture. To date, (n=43) coupons have been analyzed for spore kill-rates with regards to ice temperature and radiation exposure time. Results will be presented on the effect of cryogenic temperatures in improving radiation resistance of bacterial spores. This works also details methodology improvements by comparing different spore deposition and recovery methods before and after cryogenic irradiation.

Formaldehyde gas inactivation of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials.

PubMed

Rogers, J V; Choi, Y W; Richter, W R; Rudnicki, D C; Joseph, D W; Sabourin, C L K; Taylor, M L; Chang, J C S

2007-10-01

To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using formaldehyde gas. B. anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to approx. 1100 ppm formaldehyde gas for 10 h. Formaldehyde exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with B. subtilis (galvanized metal and painted wallboard paper) and G. stearothermophilus (industrial carpet and painted wallboard paper). Formaldehyde gas inactivated>or=50% of the biological indicators and spore strips (approx. 1x10(6) CFU) when analyzed after 1 and 7 days. Formaldehyde gas significantly reduced the number of viable spores on both porous and nonporous materials in which the two surrogates exhibited similar log reductions to that of B. anthracis on most test materials. These results provide new comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using formaldehyde gas.

Evaluating the Transport of Bacillus subtilis Spores as a Potential Surrogate for Cryptosporidium parvum Oocysts.

PubMed

Bradford, Scott A; Kim, Hyunjung; Headd, Brendan; Torkzaban, Saeed

2016-02-02

The U.S. Environmental Protection Agency has recommended the use of aerobic spores as an indicator for Cryptosporidium oocysts when determining groundwater under the direct influence of surface water. Surface properties, interaction energies, transport, retention, and release behavior of B. subtilis spores were measured over a range of physicochemical conditions, and compared with reported information for C. parvum oocysts. Interaction energy calculations predicted a much larger energy barrier and a shallower secondary minimum for spores than oocysts when the solution ionic strength (IS) equaled 0.1, 1, and 10 mM, and no energy barrier when the IS = 100 mM. Spores and oocysts exhibited similar trends of increasing retention with IS and decreasing Darcy water velocity (qw), and the predicted setback distance to achieve a six log removal was always larger for spores than oocysts. However, low levels of observed spore and oocyst release significantly influenced the predicted setback distance, especially when the fraction of reversibly retained microbes (Frev) was high. An estimate for Frev was obtained from large release pulses of spore and oocyst when the IS was reduced to deionized water. The value of Frev always increased with qw, whereas an opposition trend for Frev with IS was observed for spores (decreasing) and oocysts (increasing).

Gas discharge plasmas are effective in inactivating Bacillus and Clostridium spores.

PubMed

Tseng, Shawn; Abramzon, Nina; Jackson, James O; Lin, Wei-Jen

2012-03-01

Bacterial spores are the most resistant form of life and have been a major threat to public health and food safety. Nonthermal atmospheric gas discharge plasma is a novel sterilization method that leaves no chemical residue. In our study, a helium radio-frequency cold plasma jet was used to examine its sporicidal effect on selected strains of Bacillus and Clostridium. The species tested included Bacillus subtilis, Bacillus stearothermophilus, Clostridium sporogenes, Clostridium perfringens, Clostridium difficile, and Clostridium botulinum type A and type E. The plasmas were effective in inactivating selected Bacillus and Clostridia spores with D values (decimal reduction time) ranging from 2 to 8 min. Among all spores tested, C. botulinum type A and C. sporogenes were significantly more resistant to plasma inactivation than other species. Observations by phase contrast microscopy showed that B. subtilis spores were severely damaged by plasmas and the majority of the treated spores were unable to initiate the germination process. There was no detectable fragmentation of the DNA when the spores were treated for up to 20 min. The release of dipicolinic acid was observed almost immediately after the plasma treatment, indicating the spore envelope damage could occur quickly resulting in dipicolinic acid release and the reduction of spore resistance.

Estimating the abundance of airborne pollen and fungal spores at variable elevations using an aircraft: how high can they fly?

PubMed

Damialis, Athanasios; Kaimakamis, Evangelos; Konoglou, Maria; Akritidis, Ioannis; Traidl-Hoffmann, Claudia; Gioulekas, Dimitrios

2017-03-16

Airborne pollen and fungal spores are monitored mainly in highly populated, urban environments, for allergy prevention purposes. However, their sources can frequently be located outside cities' fringes with more vegetation. So as to shed light to this paradox, we investigated the diversity and abundance of airborne pollen and fungal spores at various environmental regimes. We monitored pollen and spores using an aircraft and a car, at elevations from sea level to 2,000 m above ground, in the region of Thesssaloniki, Greece. We found a total of 24 pollen types and more than 15 spore types. Pollen and spores were detected throughout the elevational transect. Lower elevations exhibited higher pollen concentrations in only half of plant taxa and higher fungal spore concentrations in only Ustilago. Pinaceae and Quercus pollen were the most abundant recorded by airplane (>54% of the total). Poaceae pollen were the most abundant via car measurements (>77% of the total). Cladosporium and Alternaria spores were the most abundant in all cases (aircraft: >69% and >17%, car: >45% and >27%, respectively). We conclude that pollen and fungal spores can be diverse and abundant even outside the main source area, evidently because of long-distance transport incidents.

Die another day: Fate of heat-treated Geobacillus stearothermophilus ATCC 12980 spores during storage under growth-preventing conditions.

PubMed

Mtimet, Narjes; Trunet, Clément; Mathot, Anne-Gabrielle; Venaille, Laurent; Leguérinel, Ivan; Coroller, Louis; Couvert, Olivier

2016-06-01

Geobacillus stearothermophilus spores are recognized as one of the most wet-heat resistant among aerobic spore-forming bacteria and are responsible for 35% of canned food spoilage after incubation at 55 °C. The purpose of this study was to investigate and model the fate of heat-treated survivor spores of G. stearothermophilus ATCC 12980 in growth-preventing environment. G. stearothermophilus spores were heat-treated at four different conditions to reach one or two decimal reductions. Heat-treated spores were stored in nutrient broth at different temperatures and pH under growth-preventing conditions. Spore survival during storage was evaluated by count plating over a period of months. Results reveal that G. stearothermophilus spores surviving heat treatment lose their viability during storage under growth-preventing conditions. Two different subpopulations were observed during non-thermal inactivation. They differed according to the level of their resistance to storage stress, and the proportion of each subpopulation can be modulated by heat treatment conditions. Finally, tolerance to storage stress under growth-preventing conditions increases at refrigerated temperature and neutral pH regardless of heat treatment conditions. Such results suggest that spore inactivation due to heat treatment could be completed by storage under growth-preventing conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of Bacillus atrophaeus spore viability following exposure to detonation of C4 and to deflagration of halogen-containing thermites

NASA Astrophysics Data System (ADS)

Tringe, J. W.; Létant, S. E.; Dugan, L. C.; Levie, H. W.; Kuhl, A. L.; Murphy, G. A.; Alves, S. W.; Vandersall, K. S.; Pantoya, M. L.

2013-12-01

Energetic materials are being considered for the neutralization of spore-forming bacteria. In this study, the neutralization effects of a monomolecular explosive were compared to the effects of halogen-containing thermites. Bacillus atrophaeus spores were exposed to the post-detonation environment of a 100 g charge of the military explosive C-4 at a range of 50 cm. These tests were performed in the thermodynamically closed environment of a 506-l barometric calorimeter. Associated temperatures were calculated using a thermodynamic model informed by calculations with the Cheetah thermochemical code. Temperatures in the range of 2300-2800 K were calculated to persist for nearly the full 4 ms pressure observation time. After the detonation event, spores were characterized using optical microscopy and the number of viable spores was assessed. Results showed live spore survival rates in the range of 0.01%-1%. For the thermite tests, a similar, smaller-scale configuration was employed that examined the spore neutralization effects of two thermites: aluminum with iodine pentoxide and aluminum with potassium chlorate. Only the former mixture resulted in spore neutralization. These results indicate that the detonation environment produced by an explosive with no chemical biocides may provide effective spore neutralization similar to a deflagrating thermite containing iodine.

Estimating the abundance of airborne pollen and fungal spores at variable elevations using an aircraft: how high can they fly?

NASA Astrophysics Data System (ADS)

Damialis, Athanasios; Kaimakamis, Evangelos; Konoglou, Maria; Akritidis, Ioannis; Traidl-Hoffmann, Claudia; Gioulekas, Dimitrios

2017-03-01

Airborne pollen and fungal spores are monitored mainly in highly populated, urban environments, for allergy prevention purposes. However, their sources can frequently be located outside cities’ fringes with more vegetation. So as to shed light to this paradox, we investigated the diversity and abundance of airborne pollen and fungal spores at various environmental regimes. We monitored pollen and spores using an aircraft and a car, at elevations from sea level to 2,000 m above ground, in the region of Thesssaloniki, Greece. We found a total of 24 pollen types and more than 15 spore types. Pollen and spores were detected throughout the elevational transect. Lower elevations exhibited higher pollen concentrations in only half of plant taxa and higher fungal spore concentrations in only Ustilago. Pinaceae and Quercus pollen were the most abundant recorded by airplane (>54% of the total). Poaceae pollen were the most abundant via car measurements (>77% of the total). Cladosporium and Alternaria spores were the most abundant in all cases (aircraft: >69% and >17%, car: >45% and >27%, respectively). We conclude that pollen and fungal spores can be diverse and abundant even outside the main source area, evidently because of long-distance transport incidents.

Bioforensics: Characterization of biological weapons agents by NanoSIMS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weber, P K; Ghosal, S; Leighton, T J

2007-02-26

The anthrax attacks of Fall 2001 highlight the need to develop forensic methods based on multiple identifiers to determine the origin of biological weapons agents. Genetic typing methods (i.e., DNA and RNA-based) provide one attribution technology, but genetic information alone is not usually sufficient to determine the provenance of the material. Non-genetic identifiers, including elemental and isotopic signatures, provide complementary information that can be used to identify the means, geographic location and date of production. Under LDRD funding, we have successfully developed the techniques necessary to perform bioforensic characterization with the NanoSIMS at the individual spore level. We have developedmore » methods for elemental and isotopic characterization at the single spore scale. We have developed methods for analyzing spore sections to map elemental abundance within spores. We have developed rapid focused ion beam (FIB) sectioning techniques for spores to preserve elemental and structural integrity. And we have developed a high-resolution depth profiling method to characterize the elemental distribution in individual spores without sectioning. We used these newly developed methods to study the controls on elemental abundances in spores, characterize the elemental distribution of in spores, and to study elemental uptake by spores. Our work under this LDRD project attracted FBI and DHS funding for applied purposes.« less

Bacillus subtilis Spores as Vaccine Adjuvants: Further Insights into the Mechanisms of Action

PubMed Central

de Souza, Renata Damásio; Batista, Milene Tavares; Luiz, Wilson Barros; Cavalcante, Rafael Ciro Marques; Amorim, Jaime Henrique; Bizerra, Raíza Sales Pereira; Martins, Eduardo Gimenes; de Souza Ferreira, Luís Carlos

2014-01-01

Bacillus subtilis spores have received growing attention regarding potential biotechnological applications, including the use as probiotics and in vaccine formulations. B. subtilis spores have also been shown to behave as particulate vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. In this study, we further evaluated the immune modulatory properties of B. subtilis spores using a recombinant HIV gag p24 protein as a model antigen. The adjuvant effects of B. subtilis spores were not affected by the genetic background of the mouse lineage and did not induce significant inflammatory or deleterious effects after parenteral administration. Our results demonstrated that co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen after subcutaneous administration to BALB/c and C57BL/6 mice. Spores promoted activation of antigen presenting cells as demonstrated by the upregulation of MHC and CD40 molecules and enhanced secretion of pro-inflammatory cytokines by murine dendritic cells. In addition, in vivo studies indicated a direct role of the innate immunity on the immunomodulatory properties of B. subtilis spores, as demonstrated by the lack of adjuvant effects on MyD88 and TLR2 knockout mouse strains. PMID:24475289

Modeling heat resistance of Bacillus weihenstephanensis and Bacillus licheniformis spores as function of sporulation temperature and pH.

PubMed

Baril, Eugénie; Coroller, Louis; Couvert, Olivier; Leguérinel, Ivan; Postollec, Florence; Boulais, Christophe; Carlin, Frédéric; Mafart, Pierre

2012-05-01

Although sporulation environmental factors are known to impact on Bacillus spore heat resistance, they are not integrated into predictive models used to calculate the efficiency of heating processes. This work reports the influence of temperature and pH encountered during sporulation on heat resistance of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 spores. A decrease in heat resistance (δ) was observed for spores produced either at low temperature, at high temperature or at acidic pH. Sporulation temperature and pH maximizing the spore heat resistance were identified. Heat sensitivity (z) was not modified whatever the sporulation environmental factors were. A resistance secondary model inspired by the Rosso model was proposed. Sporulation temperatures and pHs minimizing or maximizing the spore heat resistance (T(min(R)), T(opt(R)), T(max(R)), pH(min(R)) and pH(opt(R))) were estimated. The goodness of the model fit was assessed for both studied strains and literature data. The estimation of the sporulation temperature and pH maximizing the spore heat resistance is of great interest to produce spores assessing the spore inactivation in the heating processes applied by the food industry. Copyright © 2011 Elsevier Ltd. All rights reserved.

Direct high-pressure NMR observation of dipicolinic acid leaking from bacterial spore: A crucial step for thermal inactivation.

PubMed

Akasaka, Kazuyuki; Maeno, Akihiro; Yamazaki, Akira

2017-12-01

A bacterial spore protects itself with an unusually high concentration (~10% in dry weight of spore) of dipicolinic acid (DPA), the release of which is considered the crucial step for inactivating it under mild pressure and temperature conditions. However, the process of how the spore releases DPA in response to pressure remains obscure. Here we apply 1 H high-resolution high-pressure NMR spectroscopy, for the first time, to the spore suspension of Bacillus subtilis natto and monitor directly and in real-time the leaking process of DPA in response to pressure of 200MPa at 20°C. We find that about one third of the total DPA leaks immediately upon applying pressure, but that the rest leaks slowly in hrs upon decreasing the pressure. Once DPA is fully released from the spore, the proteins of the spore become easily denatured at a mild temperature, e.g., 80°C, much below the temperature commonly used to inactivate spores (121°C). The success of the present experiment opens a new avenue for studying bacterial spores and cells at the molecular level in response to pressure, temperature and other perturbations. Copyright © 2017 Elsevier B.V. All rights reserved.

Feasibility of detection and identification of individual bioaerosols using laser-induced breakdown spectroscopy.

PubMed

Dixon, P B; Hahn, D W

2005-01-15

The detection and identification of individual bioaerosols using laser-induced breakdown spectroscopy (LIBS) is investigated using aerosolized Bacillus spores. Spores of Bacillus atrophaeous, Bacillus pumilus, and Bacillus stearothemophilus were introduced into an aerosol flow stream in a prescribed manner such that single-particle LIBS detection was realized. Bacillus spores were successfully detected based on the presence of the 393.4- and 396.9-nm calcium atomic emission lines. Statistical analyses based on the aerosol number density, the LIBS-based spore sampling frequency, and the distribution of the resulting calcium mass loadings support the conclusion of individual spore detection within single-shot laser-induced plasmas. The average mass loadings were in the range of 2-3 fg of calcium/Bacillus spore, which corresponds to a calcium mass percentage of approximately 0.5%. While individual spores were detected based on calcium emission, the resulting Bacillus spectra were free from CN emission bands, which has implications for the detection of elemental carbon, and LIBS-based detection of single spores based on the presence of magnesium or sodium atomic emission was unsuccessful. Based on the current instrumental setup and analyses, real-time LIBS-based detection and identification of single Bacillus spores in ambient (i.e., real life) conditions appears unfeasible.

Estimating the abundance of airborne pollen and fungal spores at variable elevations using an aircraft: how high can they fly?

PubMed Central

Damialis, Athanasios; Kaimakamis, Evangelos; Konoglou, Maria; Akritidis, Ioannis; Traidl-Hoffmann, Claudia; Gioulekas, Dimitrios

2017-01-01

Airborne pollen and fungal spores are monitored mainly in highly populated, urban environments, for allergy prevention purposes. However, their sources can frequently be located outside cities’ fringes with more vegetation. So as to shed light to this paradox, we investigated the diversity and abundance of airborne pollen and fungal spores at various environmental regimes. We monitored pollen and spores using an aircraft and a car, at elevations from sea level to 2,000 m above ground, in the region of Thesssaloniki, Greece. We found a total of 24 pollen types and more than 15 spore types. Pollen and spores were detected throughout the elevational transect. Lower elevations exhibited higher pollen concentrations in only half of plant taxa and higher fungal spore concentrations in only Ustilago. Pinaceae and Quercus pollen were the most abundant recorded by airplane (>54% of the total). Poaceae pollen were the most abundant via car measurements (>77% of the total). Cladosporium and Alternaria spores were the most abundant in all cases (aircraft: >69% and >17%, car: >45% and >27%, respectively). We conclude that pollen and fungal spores can be diverse and abundant even outside the main source area, evidently because of long-distance transport incidents. PMID:28300143

Plate assay for determining the time of production of protease, cellulase, and pectinases by germinating fungal spores.

PubMed

Hagerman, A E; Blau, D M; McClure, A L

1985-12-01

A new method for detecting enzymes produced by fungal spores during germination is described here. With this method, the production of enzymes such as protease, cellulase, or pectinase can be correlated with the extent of spore germination. Germination is studied in vitro on agar-based media containing protein, cellulose, or pectin. The spores are immobilized on a permeable membrane mounted on the substrate-containing medium. At various times after inoculation the membrane-bound spores are removed and the medium is stained. The extent of germination is assessed by microscopic examination of the spores and the presence of active hydrolytic enzymes is revealed by the staining. The staining methods are sensitive; detection limits are 1 X 10(-3) unit of cellulase; 2 X 10(-4) unit of protease; 3 X 10(-3) unit of pectin lyase; 3.5 units of polygalacturonase; 2 X 10(-3) unit of pectin methyl esterase. The method has been demonstrated by studying the production of enzymes by germinating conidia of Botrytis cinerea. Cellulase and protease were present before any spores germinated. Pectin lyase was first observed when at least 80% of the spores had germinated. Pectin methyl esterase and polygalacturonase were not produced by the spores.

Effect of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production.

PubMed

Posada-Uribe, Luisa F; Romero-Tabarez, Magally; Villegas-Escobar, Valeska

2015-10-01

Bacillus subtilis spores have important biotechnological applications; however, achieving both, high spore cell densities and sporulation efficiencies in fermentation, is poorly reported. In this study, medium components and culture conditions were optimized with different statistical methods to increase spore production of the plant growth promoting rhizobacteria B. subtilis EA-CB0575. Key medium components were determined with Plackett-Burman (PB) design, and the optimum concentration levels of two components (glucose, MgSO4·7H2O) were optimized with a full factorial and central composite design, achieving 1.37 × 10(9) CFU/mL of spore cell density and 93.5 % of sporulation efficiency in shake flask. The optimized medium was used to determine the effect of culture conditions on spore production at bioreactor level, finding that maintaining pH control did not affect significantly spore production, while the interaction of agitation and aeration rates had a significant effect on spore cell density. The overall optimization generated a 17.2-fold increase in spore cell density (8.78 × 10(9) CFU/mL) and 1.9-fold increase in sporulation efficiency (94.2 %) compared to that of PB design. These results indicate the potential of B. subtilis EA-CB0575 to produce both, high spore cell densities and sporulation efficiencies, with very low nutrient requirements and short incubation period which can represent savings of process production.

Survival of Spores of Trichoderma longibrachiatum in Space: data from the Space Experiment SPORES on EXPOSE-R

NASA Astrophysics Data System (ADS)

Neuberger, Katja; Lux-Endrich, Astrid; Panitz, Corinna

2015-01-01

In the space experiment `Spores in artificial meteorites' (SPORES), spores of the fungus Trichoderma longibrachiatum were exposed to low-Earth orbit for nearly 2 years on board the EXPOSE-R facility outside of the International Space Station. The environmental conditions tested in space were: space vacuum at 10-7-10-4 Pa or argon atmosphere at 105 Pa as inert gas atmosphere, solar extraterrestrial ultraviolet (UV) radiation at λ > 110 nm or λ > 200 nm with fluences up to 5.8 × 108 J m-2, cosmic radiation of a total dose range from 225 to 320 mGy, and temperature fluctuations from -25 to +50°C, applied isolated or in combination. Comparable control experiments were performed on ground. After retrieval, viability of spores was analysed by two methods: (i) ethidium bromide staining and (ii) test of germination capability. About 30% of the spores in vacuum survived the space travel, if shielded against insolation. However, in most cases no significant decrease was observed for spores exposed in addition to the full spectrum of solar UV irradiation. As the spores were exposed in clusters, the outer layers of spores may have shielded the inner part. The results give some information about the likelihood of lithopanspermia, the natural transfer of micro-organisms between planets. In addition to the parameters of outer space, sojourn time in space seems to be one of the limiting parameters.

Early development of fern gametophytes in microgravity

NASA Technical Reports Server (NTRS)

Roux, Stanley J.; Chatterjee, Ani; Hillier, Sheila; Cannon, Tom

2003-01-01

Dormant spores of the fern Ceratopteris richardii were flown on Shuttle mission STS-93 to evaluate the effects of micro-g on their development and on their pattern of gene expression. Prior to flight the spores were sterilized and sown into one of two environments: (1) Microscope slides in a video-microscopy module; and (2) Petri dishes. All spores were then stored in darkness until use. Spore germination was initiated on orbit after exposure to light. For the spores on microscope slides, cell level changes were recorded through the clear spore coat of the spores by video microscopy. After their exposure to light, spores in petri dishes were frozen in orbit at four different time points during which on earth gravity fixes the polarity of their development. Spores were then stored frozen in Biological Research in Canister units until recovery on earth. The RNAs from these cells and from 1-g control cells were extracted and analyzed on earth after flight to assay changes in gene expression. Video microscopy results revealed that the germinated spores developed normally in microgravity, although the polarity of their development, which is guided by gravity on earth, was random in space. Differential Display-PCR analyses of RNA extracted from space-flown cells showed that there was about a 5% change in the pattern of gene expression between cells developing in micro-g compared to those developing on earth. c2002 Published by Elsevier Science Ltd on behalf of COSPAR.

Utilization of Low-Pressure Plasma to Inactivate Bacterial Spores on Stainless Steel Screws

PubMed Central

Stapelmann, Katharina; Fiebrandt, Marcel; Raguse, Marina; Awakowicz, Peter; Reitz, Günther

2013-01-01

Abstract A special focus area of planetary protection is the monitoring, control, and reduction of microbial contaminations that are detected on spacecraft components and hardware during and after assembly. In this study, wild-type spores of Bacillus pumilus SAFR-032 (a persistent spacecraft assembly facility isolate) and the laboratory model organism B. subtilis 168 were used to study the effects of low-pressure plasma, with hydrogen alone and in combination with oxygen and evaporated hydrogen peroxide as a process gas, on spore survival, which was determined by a colony formation assay. Spores of B. pumilus SAFR-032 and B. subtilis 168 were deposited with an aseptic technique onto the surface of stainless steel screws to simulate a spore-contaminated spacecraft hardware component, and were subsequently exposed to different plasmas and hydrogen peroxide conditions in a very high frequency capacitively coupled plasma reactor (VHF-CCP) to reduce the spore burden. Spores of the spacecraft isolate B. pumilus SAFR-032 were significantly more resistant to plasma treatment than spores of B. subtilis 168. The use of low-pressure plasma with an additional treatment of evaporated hydrogen peroxide also led to an enhanced spore inactivation that surpassed either single treatment when applied alone, which indicates the potential application of this method as a fast and suitable way to reduce spore-contaminated spacecraft hardware components for planetary protection purposes. Key Words: Bacillus spores—Contamination—Spacecraft hardware—Plasma sterilization—Planetary protection. Astrobiology 13, 597–606. PMID:23768085

One-step purification of Enterocytozoon bieneusi spores from human stools by immunoaffinity expanded-bed adsorption.

PubMed

Accoceberry, I; Thellier, M; Datry, A; Desportes-Livage, I; Biligui, S; Danis, M; Santarelli, X

2001-05-01

An original, reliable, and reproducible method for the purification of Enterocytozoon bieneusi spores from human stools is described. We recently reported the production of a species-specific monoclonal antibody (MAb) 6E52D9 immunoglobulin G2a (IgG2a) raised against the exospore of E. bieneusi spore walls. The MAb was used as a ligand to develop an immunoaffinity matrix. The mouse IgG2a MAb was bound directly to a Streamline rProtein A adsorbent, used for expanded-bed adsorption of immunoglobulins, for optimal spatial orientation of the antibody and maximum binding efficiency of the antigen. The complex was then cross-linked covalently using dimethyl pimelimidate dihydrochloride. After incubation of the immunoaffinity matrix with filtered stool samples containing numerous E. bieneusi spores and before elution with 6 M guanidine HCl, the expansion of the adsorbent bed eliminated all the fecal contaminants. The presence of spores in the elution fractions was determined by an indirect immunofluorescence antibody test (IFAT). E. bieneusi spores were found in the elution fraction in all four experiments and were still highly antigenic as indicated by IFAT. Smears examined by light microscopy contained very clean spores with no fecal debris or background bacterial and fungal contaminants. However, spore recovery rates were relatively low: an average of 10(7) spores were purified per run. This technique for isolating E. bieneusi spores directly from human stool samples with a high degree of purity opens up new approaches for studying this parasite.

A fluorescence in situ staining method for investigating spores and vegetative cells of Clostridia by confocal laser scanning microscopy and structured illuminated microscopy.

PubMed

D'Incecco, P; Ong, L; Gras, S; Pellegrino, L

2018-04-18

Non-pathogenic spore-forming Clostridia are of increasing interest due to their application in biogas production and their capability to spoil different food products. The life cycle for Clostridium includes a spore stage that can assist in survival under environmentally stressful conditions, such as extremes of temperature or pH. Due to their size, spores can be investigated by a range of microscopic techniques, many of which involve sample pre-treatment. We have developed a quick, simple and non-destructive fluorescent staining procedure that allows a clear differentiation between spores and vegetative cells and effectively stains spores, allowing recovery and tracking in subsequent experiments. Hoechst 34580, Propidium iodide and wheat germ agglutinin WGA 488 were used in combination to stain four strains of Clostridia at different life cycle stages. Staining was conducted without drying the sample, preventing changes induced by dehydration and cells observed by confocal laser scanner microscopy or using a super-resolution microscope equipped with a 3D-structured illumination module. Dual staining with Hoechst/Propidium iodide differentiated spores from vegetative cells, provided information on the viability of cells and was successfully applied to follow spore production induced by heating. Super-resolution microscopy of spores probed by Hoechst 34580 also allowed chromatin to be visualised. Direct staining of a cheese specimen using Nile Red and Fast Green allowed in situ observation of spores within the cheese and their position within the cheese matrix. The proposed staining method has broad applicability and can potentially be applied to follow Clostridium spore behaviour in a range of different environments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Oral delivery of Bacillus subtilis spore expressing enolase of Clonorchis sinensis in rat model: induce systemic and local mucosal immune responses and has no side effect on liver function.

PubMed

Yu, Jinyun; Chen, Tingjin; Xie, Zhizhi; Liang, Pei; Qu, Honglin; Shang, Mei; Mao, Qiang; Ning, Dan; Tang, Zeli; Shi, Mengchen; Zhou, Lina; Huang, Yan; Yu, Xinbing

2015-07-01

Caused by the consumption of raw or undercooked freshwater fish containing infective metacercariae of Clonorchis sinensis, human clonorchiasis remains a major public health problem in China. In previous study, we had expressed enolase from C. sinensis (CsENO) on the surface of Bacillus subtilis spore and the recombinant spore induced a pronounced protection in terms of reduced worm burden and eggs per gram feces, suggesting B. subtilis spore as an ideal vehicle for antigen delivery by oral treatment and CsENO as a promising vaccine candidate against clonorchiasis. In the current study, we detected CsENO-specific IgG and IgA levels both in serum and in intestinal mucus from rats orally administrated with B. subtilis spore surface expressing CsENO by ELISA. Lysozyme levels in serum and in intestinal mucus were analyzed too. In addition, IgA-secreting cells in intestine epithelium of the rats were detected by immunohistochemistry assay. The intestinal villi lengths of duodenum, jejunum, and ileum were also measured. Rats orally treated with B. subtilis spore or normal saline were used as controls. Our results showed that, compared with the control groups, oral administration of B. subtilis spore expressing CsENO induced both systemic and local mucosal immune response. The recombinant spores also enhanced non-specific immune response in rats. The spores had no side effect on liver function. Moreover, it might facilitate food utilization and digestion of the rats. Our work will pave the way to clarify the involved mechanisms of protective efficacy elicited by B. subtilis spore expressing CsENO and encourage us to carry out more assessment trails of the oral treated spore to develop vaccine against clonorchiasis.

Influence of temperature and organic load on chemical disinfection of Geobacillus steareothermophilus spores, a surrogate for Bacillus anthracis

PubMed Central

Guan, Jiewen; Chan, Maria; Brooks, Brian W.; Rohonczy, Liz

2013-01-01

This study evaluated the influence of temperature and organic load on the effectiveness of domestic bleach (DB), Surface Decontamination Foam (SDF), and Virkon in inactivating Geobacillus stearothermophilus spores, which are a surrogate for Bacillus anthracis spores. The spores were suspended in light or heavy organic preparations and the suspension was applied to stainless steel carrier disks. The dried spore inoculum was covered with the disinfectants and the disks were then incubated at various temperatures. At −20°C, the 3 disinfectants caused less than a 2.0 log10 reduction of spores in both organic preparations during a 24-h test period. At 4°C, the DB caused a 4.4 log10 reduction of spores in light organic preparations within 2 h, which was about 3 log10 higher than what was achieved with SDF or Virkon. In heavy organic preparations, after 24 h at 4°C the SDF had reduced the spore count by 4.5 log10, which was about 2 log10 higher than for DB or Virkon. In general, the disinfectants were most effective at 23°C but a 24-h contact time was required for SDF and Virkon to reduce spore counts in both organic preparations by at least 5.5 log10. Comparable disinfecting activity with DB only occurred with the light organic load. In summary, at temperatures as low as 4°C, DB was the most effective disinfectant, inactivating spores within 2 h on surfaces with a light organic load, whereas SDF produced the greatest reduction of spores within 24 h on surfaces with a heavy organic load. PMID:24082400

Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

NASA Technical Reports Server (NTRS)

Raghavan, V.

1991-01-01

Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.

Sampling and inactivation of wet disseminated spores from flooring materials using commercially available robotic vacuum cleaners.

PubMed

Thompson, Katy-Anne; Paton, Susan; Pottage, Thomas; Bennett, Allan

2018-05-09

Four commercially available robotic vacuum cleaners were assessed for sampling efficiency of wet disseminated Bacillus atrophaeus spores on carpet, Polyvinyl Chloride (PVC) and laminate flooring. Furthermore, their operability was evaluated and decontamination efficiency of one robot was assessed using a sodium hypochlorite solution. In an environmental chamber, robots self-navigated around 4 m 2 of flooring containing a single contaminated 0.25 m 2 tile (ca. 10 4 spores per cm 2 ). Contamination levels at pre-determined locations were assessed by macrofoam swabs (PVC and laminate) or water soluble tape (carpet), before and after sampling. Robots were dismantled post-sampling and spore recoveries assessed. Aerosol contamination was also measured during sampling. Robot sampling efficiencies were variable, however, robots recovered most spores from laminate (up to 17.1%), then PVC, and lastly carpet. All robots spread contamination from the 'hotspot' (all robots spread < 0.6% of the contamination to other areas) and became surface contaminated. Spores were detected at low levels during air sampling (<5.6 spores l -1 ). Liquid decontamination inactivated 99.1% of spores from PVC. Robotic vacuum cleaners show promise for both sampling and initial decontamination of indoor flooring. In the event of a bioterror incident, e.g. deliberate release of Bacillus anthracis spores, areas require sampling to determine the magnitude and extent of contamination, and to establish decontamination efficacy. In this study we investigate robotic sampling methods against high concentrations of bacterial spores applied by wet deposition to different floorings, contamination spread to other areas, potential transfer of spores to the operators and assessment of a wet vacuum robot for spore inactivation. The robots' usability was evaluated and how they can be employed in real life scenarios. This will help to reduce the economic cost of sampling and the risk to sampling/decontamination teams. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Effects of fungal species, cultivation time, growth substrate, and air exposure velocity on the fluorescence properties of airborne fungal spores.

PubMed

Saari, S; Mensah-Attipoe, J; Reponen, T; Veijalainen, A M; Salmela, A; Pasanen, P; Keskinen, J

2015-12-01

Real-time bioaerosol monitoring is possible with fluorescence based instruments. This study provides information on major factors that can affect the fluorescence properties of airborne fungal spores. Two fluorescence-based bioaerosol detectors, BioScout, and ultraviolet aerodynamic particle sizer (UVAPS), were used to study fluorescent particle fractions (FPFs) of released spores of three fungal species (Aspergillus versicolor, Cladosporium cladosporioides, and Penicillium brevicompactum). Two culture media (agar and gypsum board), three ages of the culture (one week, one month, and four months), and three aerosolization air velocities (5, 15, and 27 m/s) were tested. The results showed that the FPF values for spores released from gypsum were typically lower than for those released from agar indicating that poor nutrient substrate produces spores with lower amounts of fluorescent compounds. The results also showed higher FPF values with lower air velocities in aerosolization. This indicates that easily released fully developed spores have more fluorescent compounds compared to forcibly extracted non-matured spores. The FPFs typically were lower with older samples. The FPF results between the two instruments were similar, except with four-month-old samples. The results can be utilized in field measurements of fungal spores to estimate actual concentrations and compare different instruments with fluorescence-based devices as well as in instrument calibration and testing in laboratory conditions. Fluorescence-based instruments are the only choice for real-time detection of fungal spores at the moment. In general, all fluorescence-based bioaerosol instruments are tested against known bacterial and fungal spores in laboratory conditions. This study showed that fungal species, growth substrate, age of culture, and air current exposure rate have an effect on detection efficiency of fungal spores in the fluorescence-based instruments. Therefore, these factors should be considered in the instrument calibration process. The results are also important when interpreting results of fluorescence-based field measurements of fungal spores. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cleaning and Disinfection of Bacillus cereus Biofilm.

PubMed

Deal, Amanda; Klein, Dan; Lopolito, Paul; Schwarz, John Spencer

2016-01-01

Methodology has been evolving for the testing of disinfectants against bacterial single-species biofilms, as the difficulty of biofilm remediation continues to gain much-needed attention. Bacterial single-species biofilm contamination presents a real risk to good manufacturing practice-regulated industries. However, mixed-species biofilms and biofilms containing bacterial spores remain an even greater challenge for cleaning and disinfection. Among spore-forming microorganisms frequently encountered in pharmaceutical manufacturing areas, the spores of Bacillus cereus are often determined to be the hardest to disinfect and eradicate. One of the reasons for the low degree of susceptibility to disinfection is the ability of these spores to be encapsulated within an exopolysachharide biofilm matrix. In this series of experiments, we evaluated the disinfectant susceptibility of B. cereus biofilms relative to disassociated B. cereus spores and biofilm from a non-spore-forming species. Further, we assessed the impact that pre-cleaning has on increasing that susceptibility. Methodology has been evolving for the testing of disinfectants against bacterial single-species biofilms, as the difficulty of biofilm remediation continues to gain much-needed attention. Bacterial single-species biofilm contamination presents a real risk to good manufacturing practice-regulated industries. However, mixed-species biofilms and biofilms containing bacterial spores remain an even greater challenge for cleaning and disinfection. Among spore-forming microorganisms frequently encountered in pharmaceutical manufacturing areas, the spores of Bacillus cereus are often determined to be the hardest to disinfect and eradicate. One of the reasons for the low degree of susceptibility to disinfection is the ability of these spores to be encapsulated within an exopolysachharide biofilm matrix. In this series of experiments, we evaluated the disinfectant susceptibility of B. cereus biofilms relative to disassociated B. cereus spores and biofilm from a non-spore-forming species. Further, we assessed the impact that pre-cleaning has on increasing that susceptibility. © PDA, Inc. 2016.

Chemical and Stress Resistances of Clostridium difficile Spores and Vegetative Cells

PubMed Central

Edwards, Adrianne N.; Karim, Samiha T.; Pascual, Ricardo A.; Jowhar, Lina M.; Anderson, Sarah E.; McBride, Shonna M.

2016-01-01

Clostridium difficile is a Gram-positive, sporogenic and anaerobic bacterium that causes a potentially fatal colitis. C. difficile enters the body as dormant spores that germinate in the colon to form vegetative cells that secrete toxins and cause the symptoms of infection. During transit through the intestine, some vegetative cells transform into spores, which are more resistant to killing by environmental insults than the vegetative cells. Understanding the inherent resistance properties of the vegetative and spore forms of C. difficile is imperative for the development of methods to target and destroy the bacterium. The objective of this study was to define the chemical and environmental resistance properties of C. difficile vegetative cells and spores. We examined vegetative cell and spore tolerances of three C. difficile strains, including 630Δerm, a 012 ribotype and a derivative of a past epidemic strain; R20291, a 027 ribotype and current epidemic strain; and 5325, a clinical isolate that is a 078 ribotype. All isolates were tested for tolerance to ethanol, oxygen, hydrogen peroxide, butanol, chloroform, heat and sodium hypochlorite (household bleach). Our results indicate that 630Δerm vegetative cells (630 spo0A) are more resistant to oxidative stress than those of R20291 (R20291 spo0A) and 5325 (5325 spo0A). In addition, 5325 spo0A vegetative cells exhibited greater resistance to organic solvents. In contrast, 630Δerm spores were more sensitive than R20291 or 5325 spores to butanol. Spores from all three strains exhibited high levels of resistance to ethanol, hydrogen peroxide, chloroform and heat, although R20291 spores were more resistant to temperatures in the range of 60–75°C. Finally, household bleach served as the only chemical reagent tested that consistently reduced C. difficile vegetative cells and spores of all tested strains. These findings establish conditions that result in vegetative cell and spore elimination and illustrate the resistance of C. difficile to common decontamination methods. These results further demonstrate that the vegetative cells and spores of various C. difficile strains have different resistance properties that may impact decontamination of surfaces and hands. PMID:27833595

Ice Nucleation of Fungal Spores from the Classes Agaricomycetes, Ustilaginomycetes, and Eurotiomycetes, and the effect on the Atmospheric Transport of these Spores

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haga, D. I.; Burrows, Susannah M.; Iannone, R.

2014-08-26

Ice nucleation on fungal spores may affect the frequency and properties of ice and mixed-phase clouds. We studied the ice nucleation properties of 12 different species of fungal spores chosen from three classes: Agaricomycetes, Ustilagomycetes, and Eurotiomycetes. Agaricomycetes include many types of mushroom species and are cosmopolitan all over the globe. Ustilagomycetes are agricultural pathogens and have caused widespread damage to crops. Eurotiomycetes are found on all types of decaying material and include important human allergens. We focused on these classes since they are thought to be abundant in the atmosphere and because there is very little information on themore » ice nucleation ability of these classes of spores in the literature. All of the fungal spores investigated were found to cause freezing of water droplets at temperatures warmer than homogeneous freezing. The cumulative number of ice nuclei per spore was 0.001 at temperatures between -19 °C and -29 °C, 0.01 between -25.5 °C and -31 °C, and 0.1 between -26 °C and -36 °C. On average, the order of ice nucleating ability for these spores is Ustilagomycetes > Agaricomycetes ≅ Eurotiomycetes. We show that at temperatures below -20 °C, all of the fungal spores studied here are less efficient ice nuclei compared to Asian mineral dust on a per surface area basis. We used our new freezing results together with data in the literature to compare the freezing temperatures of spores from the phyla Basidiomycota and Ascomycota, which together make up 98 % of known fungal species found on Earth. The data show that within both phyla (Ascomycota and Basidiomycota) there is a wide range of freezing properties, and also that the variation within a phylum is greater than the variation between the average freezing properties of the phyla. Using a global chemistry-climate transport model, we investigated whether ice nucleation on the studied spores, followed by precipitation, can influence the atmospheric transport and global distributions of these spores in the atmosphere. Simulations show that inclusion of ice nucleation scavenging of fungal spores in mixed-phase clouds can decrease the surface annual mean mixing ratios of fungal spores over the oceans and polar regions and decrease annual mean mixing ratios in the upper troposphere.« less

Autoclave performance and practitioner knowledge of autoclave use: a survey of selected UK practices.

PubMed

Burke, F J; Coulter, W A; Cheung, S W; Palenik, C J

1998-04-01

It is essential that the dental surgery autoclave be tested regularly to ensure that it is operating at a temperature and pressure that will kill all microorganisms. The aims of this study were to investigate the knowledge of general dental practitioners on aspects of autoclave use, to examine their autoclaving routines, and to test the effectiveness of the sterilization cycle of their practice's autoclave. A 35-question questionnaire was distributed to 840 dental practitioners in the United Kingdom. They were also sent three spore test ampules with details of their function and instructions on their use. The practitioners were asked to use the spore test ampules in their own practice's autoclave and to return these for testing. Four hundred one questionnaires and associated spore test ampules were returned for analysis, a 47.7% response rate. Eight practice autoclaves (2.0%) failed to sterilize the spores. These practices were offered a retest following counseling. A total of 77.8% of respondents had received formal training in infection control. Of the 71.3% of respondents who stated that they check the performance of their autoclave routinely, 31.5% use a chemical strip every cycle, 17.5% use it once per day, and 30.4% use it once per week. However, only five respondents (1.2%) use a spore test; two of these respondents use it every cycle, one once per day, and two once per week. Further education of practitioners may be necessary to enhance their awareness of the need for routine checking of autoclave effectiveness.

14C Analysis of protein extracts from Bacillus spores.

PubMed

Cappuccio, Jenny A; Falso, Miranda J Sarachine; Kashgarian, Michaele; Buchholz, Bruce A

2014-07-01

Investigators of bioagent incidents or interdicted materials need validated, independent analytical methods that will allow them to distinguish between recently made bioagent samples versus material drawn from the archives of a historical program. Heterotrophic bacteria convert the carbon in their food sources, growth substrate or culture media, into the biomolecules they need. The F(14)C (fraction modern radiocarbon) of a variety of media, Bacillus spores, and separated proteins from Bacillus spores was measured by accelerator mass spectrometry (AMS). AMS precisely measures F(14)C values of biological materials and has been used to date the synthesis of biomaterials over the bomb pulse era (1955 to present). The F(14)C of Bacillus spores reflects the radiocarbon content of the media in which they were grown. In a survey of commercial media we found that the F(14)C value indicated that carbon sources for the media were alive within about a year of the date of manufacture and generally of terrestrial origin. Hence, bacteria and their products can be dated using their (14)C signature. Bacillus spore samples were generated onsite with defined media and carbon free purification and also obtained from archived material. Using mechanical lysis and a variety of washes with carbon free acids and bases, contaminant carbon was removed from soluble proteins to enable accurate (14)C bomb-pulse dating. Since media is contemporary, (14)C bomb-pulse dating of isolated soluble proteins can be used to distinguish between historical archives of bioagents and those produced from recent media. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

14C Analysis of Protein Extracts from Bacillus Spores

PubMed Central

Cappucio, Jenny A.; Sarachine Falso, Miranda J.; Kashgarian, Michaele; Buchholz, Bruce A.

2014-01-01

Investigators of bioagent incidents or interdicted materials need validated, independent analytical methods that will allow them to distinguish between recently made bioagent samples versus material drawn from the archives of a historical program. Heterotrophic bacteria convert the carbon in their food sources, growth substrate or culture media, into the biomolecules they need. The F14C (fraction modern radiocarbon) of a variety of media, Bacillus spores, and separated proteins from Bacillus spores was measured by accelerator mass spectrometry (AMS). AMS precisely measures F14C values of biological materials and has been used to date the synthesis of biomaterials over the bomb pulse era (1955 to present). The F14C of Bacillus spores reflects the radiocarbon content of the media in which they were grown. In a survey of commercial media we found that the F14C value indicated that carbon sources for the media were alive within about a year of the date of manufacture and generally of terrestrial origin. Hence, bacteria and their products can be dated using their 14C signature. Bacillus spore samples were generated onsite with defined media and carbon free purification and also obtained from archived material. Using mechanical lysis and a variety of washes with carbon free acids and bases, contaminant carbon was removed from soluble proteins to enable accurate 14C bomb-pulse dating. Since media is contemporary, 14C bomb-pulse dating of isolated soluble proteins can be used to distinguish between historical archives of bioagents and those produced from recent media. PMID:24814329

Preliminary report on the biological effects of space flight on the producing strain of a new immunosuppressant, Kanglemycin C.

PubMed

Zhou, Jianqin; Sun, Chenghang; Wang, Nanjin; Gao, Rongmei; Bai, Shuoke; Zheng, Huanrong; You, Xuefu; Li, Rongfeng

2006-08-01

Kanglemycin C (K-C) is a new immunosuppressant isolated from the culture broth of Nocardia mediterranei var. kanglensis 1747-64. To improve the productivity of K-C and to study the biological effects of space flight on its producing strain, spores from five K-C producing strains (U-10, U-15, U-7, M-13, gamma-33) mutated from the wild strain N. mediterranei var. kanglensis 1747-64 were carried into space by an unmanned spaceship, "Shenzhou III" (Divine Vessel III) on March 25, 2002. Comparatively, the strain U-7 was the highest K-C producing strain among the above five starting strains when cultivated in 500-ml Erlenmeyer flasks. After a 6 day and 18 h flight, the treated spores went through serial screening processes to screen for high-yield K-C mutant strains, using thin layer chromatography and high performance liquid chromatography (HPLC). The K-C yield produced by one mutant strain, designated as F-16, derived from the starting strain U-7 was increased by up to 200% when compared to that produced by the starting strain U-7 in 500-ml Erlenmeyer flasks after careful postflight HPLC analysis. Another mutant strain, designated as F-210, derived from the starting strain M-13 showed reduced productivity of K-C as well as exhibited changes in some morphological and physiological characteristics. For example, the broth color of the strain F-210 changed from yellow to purple after 96 h of culture, but that of the ground control strain M-13 remained yellow. Similarly, the mycelium morphological change from filamentous to coccoid of F-210 occurred later than that of ground control M-13. Examination of the survivability of postflight spores indicated that exposure to radiation, during the 162 h of space flight, plays a critical role in the survival rates of spores such that spores exposed to strong radiation exhibited lower survival rates than spores exposed to weak radiation.

Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-specific genes.

PubMed

Galperin, Michael Y; Mekhedov, Sergei L; Puigbo, Pere; Smirnov, Sergey; Wolf, Yuri I; Rigden, Daniel J

2012-11-01

Three classes of low-G+C Gram-positive bacteria (Firmicutes), Bacilli, Clostridia and Negativicutes, include numerous members that are capable of producing heat-resistant endospores. Spore-forming firmicutes include many environmentally important organisms, such as insect pathogens and cellulose-degrading industrial strains, as well as human pathogens responsible for such diseases as anthrax, botulism, gas gangrene and tetanus. In the best-studied model organism Bacillus subtilis, sporulation involves over 500 genes, many of which are conserved among other bacilli and clostridia. This work aimed to define the genomic requirements for sporulation through an analysis of the presence of sporulation genes in various firmicutes, including those with smaller genomes than B. subtilis. Cultivable spore-formers were found to have genomes larger than 2300 kb and encompass over 2150 protein-coding genes of which 60 are orthologues of genes that are apparently essential for sporulation in B. subtilis. Clostridial spore-formers lack, among others, spoIIB, sda, spoVID and safA genes and have non-orthologous displacements of spoIIQ and spoIVFA, suggesting substantial differences between bacilli and clostridia in the engulfment and spore coat formation steps. Many B. subtilis sporulation genes, particularly those encoding small acid-soluble spore proteins and spore coat proteins, were found only in the family Bacillaceae, or even in a subset of Bacillus spp. Phylogenetic profiles of sporulation genes, compiled in this work, confirm the presence of a common sporulation gene core, but also illuminate the diversity of the sporulation processes within various lineages. These profiles should help further experimental studies of uncharacterized widespread sporulation genes, which would ultimately allow delineation of the minimal set(s) of sporulation-specific genes in Bacilli and Clostridia. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

Auxosporulation in Paralia guyana MacGillivary (Bacillariophyta) and Possible New Insights into the Habit of the Earliest Diatoms

PubMed Central

Kaczmarska, Irena; Ehrman, James M.

2015-01-01

Background Diatoms are one of the most ecologically important aquatic micro-eukaryotes. As a group unambiguously recognized as diatoms, they seem to have appeared relatively recently with a limited record of putative remains from oldest sediments. In contrast, molecular clock estimates for the earliest possible emergence of diatoms suggest a considerably older date. Depending on the analysis, Paralia and Leptocylindrus have been recovered within the basal molecular divergences of diatoms. Thus these genera may be in the position to inform on characters that the earliest diatoms possessed. Findings Here we present auxospore development and structure of initial and post-auxospore cells in a representative of the ancient non-polar centric genus Paralia. Their initial frustules showed unusual, but not unprecedented, spore-like morphology. Similarly, initial frustules of Leptocylindrus have been long considered resting spores and a unique peculiarity of this genus. However, even though spore-like in appearance, initial cells of Paralia readily resumed mitotic divisions. In addition, Paralia post-auxospore cells underwent several rounds of mitoses in a multi-step process of building a typical, “perfect” vegetative valve. This degree of heteromorphy immediately post-auxosporulation is thus far unknown among the diatoms. Implications A spore-related origin of diatoms has already been considered, most recently in the form of the “multiplate diploid cyst” hypothesis. Our discovery that the initial cells in some of the most ancient diatom lineages are structurally spore-like is consistent with that hypothesis because the earliest diatoms may be expected to look somewhat similar to their ancestors. We speculate that because the earliest diatoms may have appeared less diatom-like and more spore-like, they could have gone unrecognized as such in the Triassic/Jurassic sediments. If correct, diatoms may indeed be much older than the fossil record indicates, and possibly more in line with some molecular clock predictions. PMID:26485144

Predominance of Viable Spore-Forming Piezophilic Bacteria in High-Pressure Enrichment Cultures from ~1.5 to 2.4 km-Deep Coal-Bearing Sediments below the Ocean Floor

PubMed Central

Fang, Jiasong; Kato, Chiaki; Runko, Gabriella M.; Nogi, Yuichi; Hori, Tomoyuki; Li, Jiangtao; Morono, Yuki; Inagaki, Fumio

2017-01-01

Phylogenetically diverse microorganisms have been observed in marine subsurface sediments down to ~2.5 km below the seafloor (kmbsf). However, very little is known about the pressure-adapted and/or pressure-loving microorganisms, the so called piezophiles, in the deep subseafloor biosphere, despite that pressure directly affects microbial physiology, metabolism, and biogeochemical processes of carbon and other elements in situ. In this study, we studied taxonomic compositions of microbial communities in high-pressure incubated sediment, obtained during the Integrated Ocean Drilling Program (IODP) Expedition 337 off the Shimokita Peninsula, Japan. Analysis of 16S rRNA gene-tagged sequences showed that members of spore-forming bacteria within Firmicutes and Actinobacteria were predominantly detected in all enrichment cultures from ~1.5 to 2.4 km-deep sediment samples, followed by members of Proteobacteria, Acidobacteria, and Bacteroidetes according to the sequence frequency. To further study the physiology of the deep subseafloor sedimentary piezophilic bacteria, we isolated and characterized two bacterial strains, 19R1-5 and 29R7-12, from 1.9 and 2.4 km-deep sediment samples, respectively. The isolates were both low G+C content, gram-positive, endospore-forming and facultative anaerobic piezophilic bacteria, closely related to Virgibacillus pantothenticus and Bacillus subtilis within the phylum Firmicutes, respectively. The optimal pressure and temperature conditions for growth were 20 MPa and 42°C for strain 19R1-5, and 10 MPa and 43°C for strain 29R7-12. Bacterial (endo)spores were observed in both the enrichment and pure cultures examined, suggesting that these piezophilic members were derived from microbial communities buried in the ~20 million-year-old coal-bearing sediments after the long-term survival as spores and that the deep biosphere may host more abundant gram-positive spore-forming bacteria and their spores than hitherto recognized. PMID:28220112

Predominance of Viable Spore-Forming Piezophilic Bacteria in High-Pressure Enrichment Cultures from ~1.5 to 2.4 km-Deep Coal-Bearing Sediments below the Ocean Floor.

PubMed

Fang, Jiasong; Kato, Chiaki; Runko, Gabriella M; Nogi, Yuichi; Hori, Tomoyuki; Li, Jiangtao; Morono, Yuki; Inagaki, Fumio

2017-01-01

Phylogenetically diverse microorganisms have been observed in marine subsurface sediments down to ~2.5 km below the seafloor (kmbsf). However, very little is known about the pressure-adapted and/or pressure-loving microorganisms, the so called piezophiles, in the deep subseafloor biosphere, despite that pressure directly affects microbial physiology, metabolism, and biogeochemical processes of carbon and other elements in situ . In this study, we studied taxonomic compositions of microbial communities in high-pressure incubated sediment, obtained during the Integrated Ocean Drilling Program (IODP) Expedition 337 off the Shimokita Peninsula, Japan. Analysis of 16S rRNA gene-tagged sequences showed that members of spore-forming bacteria within Firmicutes and Actinobacteria were predominantly detected in all enrichment cultures from ~1.5 to 2.4 km-deep sediment samples, followed by members of Proteobacteria, Acidobacteria, and Bacteroidetes according to the sequence frequency. To further study the physiology of the deep subseafloor sedimentary piezophilic bacteria, we isolated and characterized two bacterial strains, 19R1-5 and 29R7-12, from 1.9 and 2.4 km-deep sediment samples, respectively. The isolates were both low G+C content, gram-positive, endospore-forming and facultative anaerobic piezophilic bacteria, closely related to Virgibacillus pantothenticus and Bacillus subtilis within the phylum Firmicutes, respectively. The optimal pressure and temperature conditions for growth were 20 MPa and 42°C for strain 19R1-5, and 10 MPa and 43°C for strain 29R7-12. Bacterial (endo)spores were observed in both the enrichment and pure cultures examined, suggesting that these piezophilic members were derived from microbial communities buried in the ~20 million-year-old coal-bearing sediments after the long-term survival as spores and that the deep biosphere may host more abundant gram-positive spore-forming bacteria and their spores than hitherto recognized.

False Negative Rates of a Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates via Real-Time PCR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hutchison, Janine R.; Piepel, Gregory F.; Amidan, Brett G.

Surface sampling for Bacillus anthracis spores has traditionally relied on detection via bacterial cultivation methods. Although effective, this approach does not provide the level of organism specificity that can be gained through molecular techniques. False negative rates (FNR) and limits of detection (LOD) were determined for two B. anthracis surrogates with modified rapid viability-polymerase chain reaction (mRV-PCR) following macrofoam-swab sampling. This study was conducted in parallel with a previously reported study that analyzed spores using a plate-culture method. B. anthracis Sterne (BAS) or B. atrophaeus Nakamura (BG) spores were deposited onto four surface materials (glass, stainless steel, vinyl tile, andmore » plastic) at nine target concentrations (2 to 500 spores/coupon; 0.078 to 19.375 colony-forming units [CFU] per cm2). Mean FNR values for mRV-PCR analysis ranged from 0 to 0.917 for BAS and 0 to 0.875 for BG and increased as spore concentration decreased (over the concentrations investigated) for each surface material. FNRs based on mRV-PCR data were not statistically different for BAS and BG, but were significantly lower for glass than for vinyl tile. FNRs also tended to be lower for the mRV-PCR method compared to the culture method. The mRV-PCR LOD95 was lowest for glass (0.429 CFU/cm2 with BAS and 0.341 CFU/cm2 with BG) and highest for vinyl tile (0.919 CFU/cm2 with BAS and 0.917 CFU/cm2 with BG). These mRV-PCR LOD95 values were lower than the culture values (BAS: 0.678 to 1.023 CFU/cm2 and BG: 0.820 to 1.489 CFU/cm2). The FNR and LOD95 values reported in this work provide guidance for environmental sampling of Bacillus spores at low concentrations.« less

False Negative Rates of a Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates via Real-Time PCR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hutchison, Janine R.; Piepel, Gregory F.; Amidan, Brett G.

Surface sampling for Bacillus anthracis spores has traditionally relied on detection via bacterial cultivation methods. Although effective, this approach does not provide the level of organism specificity that can be gained through molecular techniques. False negative rates (FNR) and limits of detection (LOD) were determined for two B. anthracis surrogates with modified rapid viability-polymerase chain reaction (mRV-PCR) following macrofoam-swab sampling. This study was conducted in parallel with a previously reported study that analyzed spores using a plate-culture method. B. anthracis Sterne (BAS) or B. atrophaeus Nakamura (BG) spores were deposited onto four surface materials (glass, stainless steel, vinyl tile, andmore » plastic) at nine target concentrations (2 to 500 spores/coupon; 0.078 to 19.375 colony-forming units [CFU] per cm²). Mean FNR values for mRV-PCR analysis ranged from 0 to 0.917 for BAS and 0 to 0.875 for BG and increased as spore concentration decreased (over the concentrations investigated) for each surface material. FNRs based on mRV-PCR data were not statistically different for BAS and BG, but were significantly lower for glass than for vinyl tile. FNRs also tended to be lower for the mRV-PCR method compared to the culture method. The mRV-PCR LOD₉₅ was lowest for glass (0.429 CFU/cm² with BAS and 0.341 CFU/cm² with BG) and highest for vinyl tile (0.919 CFU/cm² with BAS and 0.917 CFU/cm² with BG). These mRV-PCR LOD₉₅ values were lower than the culture values (BAS: 0.678 to 1.023 CFU/cm² and BG: 0.820 to 1.489 CFU/cm²). The FNR and LOD₉₅ values reported in this work provide guidance for environmental sampling of Bacillus spores at low concentrations.« less

Bacterial spore survival after exposure to HZE particle bombardment -implication for the lithopanspermia hypothesis.

NASA Astrophysics Data System (ADS)

Moeller, Ralf; Berger, Thomas; Matthiä, Daniel; Okayasu, Ryuichi; Kitamura, H.; Reitz, Guenther

Based on their unique resistance to various space parameters, bacterial spores (mainly spores of Bacillus subtilis) are one of the model systems used for astrobiological studies. More re-cently, spores of B. subtilis have been applied for experimental research on the likelihood of interplanetary transfer of life. Since its first postulation by Arrhenius in 1903, the pansper-mia hypothesis has been revisited many-times, e.g. after the discovery of several lunar and Martian meteorites on Earth [1,2]. These information provided intriguing evidence that rocks may naturally be transferred between the terrestrial planets. The scenario of panspermia, now termed "lithopanspermia" involves three basic hypothetical steps: (i) the escape process, i.e. removal to space of biological material, which has survived being lifted from the surface to high altitudes; (ii) interim state in space, i.e., survival of the biological material over time scales comparable with interplanetary or interstellar passage; (iii) the entry process, i.e. nondestruc-tive deposition of the biological material on another planet [2]. In our research, spores of B. subtilis were used to study the effects of galactic cosmic radiation on spore survival and induced mutations. On an interplanetary journey, outside a protective magnetic field, spore-containing rocks would be exposed to bombardment by high-energy charged particle radiation from galac-tic sources and from the sun. Air-dried spore layers on three different host materials (i.e., non-porous igneous rocks (gabbro), quartz, and spacecraft analog material (aluminum)) were irradiated with accelerated heavy ions (Helium and Iron) with a LET (linear energy transfer) ˆ of 2 and 200 keV/Am, at the Heavy Ion Medical Accelerator (HIMAC) at the National In-stitute of Radiological Sciences, (NIRS), Chiba, Japan in the frame of the HIMAC research project 20B463 "Characterization of heavy ion-induced damage in Bacillus subtilis spores and their global transcriptional response during spore germination" (Moeller et al., 2008 [3]). To simulate the interplanetary journey of a meteorite, stacks of spore-samples on gabbro slides in different depths were exposed. Spore survival and the rate of the induced mutations (i.e., sporulation-deficiency (Spo-)) depended on the LET of the applied species of ions as well as on the location (and depth) of the irradiated spores in the artificial meteorite. The exposure to high LET iron ions led to a low level of spore survival and increased frequency of mutation to Spo-compared to low-energy charged particles compared to the low LET helium ions. In order to obtain insights on the role of DNA repair by nonhomologous end joining (NHEJ), homologous recombination (HR) and apurinic/apyrimidinic (AP) endonucleases in B. subtilis spore resistance to high-energy charged particles has been studied in parallel. Spores deficient in NHEJ and AP endonucleases were significantly more sensitive to HZE particle bombardment than were the HR-mutant and wild-type spores, indicating that NHEJ and AP endonucleases provide DNA break repair pathways during spore germination. ((References: [1] Arrhenius, S. 1903. Die Verbreitung des Lebens im Weltenraum. Umschau 7:481-485.; [2] Nicholson, W. L. 2009. Ancient micronauts: interplanetary transport of microbes by cosmic impacts. Trends Mi-crobiol. 17:243-250.; [3] Moeller, R., P. Setlow, G. Horneck, T. Berger, G. Reitz, P. Rettberg, A. J. Doherty, R. Okayasu, and W. L. Nicholson. 2008. Roles of the major, small, acid-soluble spore proteins and spore-specific and universal DNA repair mechanisms in resistance of Bacillus subtilis spores to ionizing radiation from X-rays and high-energy charged-particle bombardment. J. Bacteriol. 190:1134-1140.))

Microbial Inactivation for Safe and Rapid Diagnostics of Infectious Samples ▿ †

PubMed Central

Sagripanti, Jose-Luis; Hülseweh, Birgit; Grote, Gudrun; Voß, Luzie; Böhling, Katrin; Marschall, Hans-Jürgen

2011-01-01

The high risk associated with biological threat agents dictates that any suspicious sample be handled under strict surety and safety controls and processed under high-level containment in specialized laboratories. This study attempted to find a rapid, reliable, and simple method for the complete inactivation of a wide range of pathogens, including spores, vegetative bacteria, and viruses, while preserving microbial nucleic acid fragments suitable for PCRs and proteinaceous epitopes for detection by immunoassays. Formaldehyde, hydrogen peroxide, and guanidium thiocyanate did not completely inactivate high titers of bacterial spores or viruses after 30 min at 21°C. Glutaraldehyde and sodium hypochlorite showed high microbicidal activity but obliterated the PCR or enzyme-linked immunosorbent assay (ELISA) detection of bacterial spores or viruses. High-level inactivation (more than 6 log10) of bacterial spores (Bacillus atrophaeus), vegetative bacteria (Pseudomonas aeruginosa), an RNA virus (the alphavirus Pixuna virus), or a DNA virus (the orthopoxvirus vaccinia virus) was attained within 30 min at 21°C by treatment with either peracetic acid or cupric ascorbate with minimal hindrance of subsequent PCR tests and immunoassays. The data described here should provide the basis for quickly rendering field samples noninfectious for further analysis under lower-level containment and considerably lower cost. PMID:21856830

Evolution of gilled mushrooms and puffballs inferred from ribosomal DNA sequences

PubMed Central

Hibbett, David S.; Pine, Elizabeth M.; Langer, Ewald; Langer, Gitta; Donoghue, Michael J.

1997-01-01

Homobasidiomycete fungi display many complex fruiting body morphologies, including mushrooms and puffballs, but their anatomical simplicity has confounded efforts to understand the evolution of these forms. We performed a comprehensive phylogenetic analysis of homobasidiomycetes, using sequences from nuclear and mitochondrial ribosomal DNA, with an emphasis on understanding evolutionary relationships of gilled mushrooms and puffballs. Parsimony-based optimization of character states on our phylogenetic trees suggested that strikingly similar gilled mushrooms evolved at least six times, from morphologically diverse precursors. Approximately 87% of gilled mushrooms are in a single lineage, which we call the “euagarics.” Recently discovered 90 million-year-old fossil mushrooms are probably euagarics, suggesting that (i) the origin of this clade must have occurred no later than the mid-Cretaceous and (ii) the gilled mushroom morphology has been maintained in certain lineages for tens of millions of years. Puffballs and other forms with enclosed spore-bearing structures (Gasteromycetes) evolved at least four times. Derivation of Gasteromycetes from forms with exposed spore-bearing structures (Hymenomycetes) is correlated with repeated loss of forcible spore discharge (ballistospory). Diverse fruiting body forms and spore dispersal mechanisms have evolved among Gasteromycetes. Nevertheless, it appears that Hymenomycetes have never been secondarily derived from Gasteromycetes, which suggests that the loss of ballistospory has constrained evolution in these lineages. PMID:9342352