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Sample records for bacillus spores f-spores

  1. Hydrazine inactivates bacillus spores

    Science.gov (United States)

    Schubert, Wayne; Plett, G. A.; Yavrouian, A. H.; Barengoltz, J.

    2005-01-01

    Planetary Protection places requirements on the maximum number of viable bacterial spores that may be delivered by a spacecraft to another solar system body. Therefore, for such space missions, the spores that may be found in hydrazine are of concern. A proposed change in processing procedures that eliminated a 0.2 um filtration step propmpted this study to ensure microbial contamination issue existed, especially since no information was found in the literature to substantiate bacterial spore inactivation by hydrazine.

  2. Hydrazine vapor inactivates Bacillus spores

    Science.gov (United States)

    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.

  3. Combined high pressure and thermal processing on inactivation of type E and nonproteolytic type B and F spores of Clostridium botulinum.

    Science.gov (United States)

    Skinner, Guy E; Marshall, Kristin M; Morrissey, Travis R; Loeza, Viviana; Patazca, Eduardo; Reddy, N Rukma; Larkin, John W

    2014-12-01

    The aim of this study was to determine the resistance of multiple strains of the three nonproteolytic types of Clostridium botulinum (seven strains of type E, eight of type B, and two of type F) spores exposed to combined high pressure and thermal processing. The resistance of spores suspended in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7) was determined at a process temperature of 80°C with high pressures of 600, 650, and 700 MPa using a laboratory-scale pressure test system. Spores of C. botulinum serotype E strains demonstrated less resistance than nonproteolytic spores of type B or F strains when processed at 80°C and 600 MPa for up to 15 min. All C. botulinum type E strains were reduced by . 6.0 log units within 5 min under these conditions. Among the nonproteolytic type B strains, KAP 9-B was the most resistant, resulting in reductions of 2.7, 5.3, and 5.5 log, coinciding with D-values of 7.7, 3.4, and 1.8 min at 80°C and 600, 650, and 700 MPa, respectively. Of the two nonproteolytic type F strains, 610F was the most resistant, showing 2.6-, 4.5-, and 5.3-log reductions with D-values of 8.9, 4.3, and 1.8 min at 80°C and 600, 650, and 700 MPa, respectively. Pulsed-field gel electrophoresis was performed to examine the genetic relatedness of strains tested and to determine if strains with similar banding patterns also exhibited similar D-values. No correlation between the genetic fingerprint of a particular strain and its resistance to high pressure processing was observed.

  4. Measurement of Metabolic Activity in Dormant Spores of Bacillus Species

    Science.gov (United States)

    2015-01-14

    SECURITY CLASSIFICATION OF: Spores of Bacillus megaterium and Bacillus subtilis were harvested shortly after release from sporangia, incubated under...Dec-2014 Approved for Public Release; Distribution Unlimited Final Report: Measurement of Metabolic Activity in Dormant Spores of Bacillus Species...Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 spores, Bacillus , spore dormancy, 3-phosphoglycerate REPORT DOCUMENTATION PAGE 11

  5. Quantitative immunofluorescence studies of the serology of Bacillus anthracis spores.

    OpenAIRE

    1983-01-01

    A fluorescein-conjugated antibody against formalin-inactivated spores of Bacillus anthracis Vollum reacted only weakly with a variety of Bacillus species in microfluorometric immunofluorescence assays. A conjugated antibody against spores of B. anthracis Sterne showed little affinity for spores of several B. anthracis isolates including B. anthracis Vollum, indicating that more than one anthrax spore serotype exists.

  6. Nano-Mechanical Properties of Heat Inactivated Bacillus anthracis and Bacillus thuringiensis Spores

    Science.gov (United States)

    2008-03-01

    NANO-MECHANICAL PROPERTIES OF HEAT INACTIVATED BACILLUS ANTHRACIS AND BACILLUS THURINGIENSIS ...GAP/ENP/08-M07 NANO-MECHANICAL PROPERTIES OF HEAT INACTIVATED BACILLUS ANTHRACIS AND BACILLUS THURINGIENSIS SPORES THESIS...AFIT/GAP/ENP/08-M07 NANO-MECHANICAL PROPERTIES OF HEAT INACTIVATED BACILLUS ANTHRACIS AND BACILLUS THURINGIENSIS SPORES Jessica

  7. Methyl Iodide Fumigation of Bacillus anthracis Spores.

    Science.gov (United States)

    Sutton, Mark; Kane, Staci R; Wollard, Jessica R

    2015-09-01

    Fumigation techniques such as chlorine dioxide, vaporous hydrogen peroxide, and paraformaldehyde previously used to decontaminate items, rooms, and buildings following contamination with Bacillus anthracis spores are often incompatible with materials (e.g., porous surfaces, organics, and metals), causing damage or residue. Alternative fumigation with methyl bromide is subject to U.S. and international restrictions due to its ozone-depleting properties. Methyl iodide, however, does not pose a risk to the ozone layer and has previously been demonstrated as a fumigant for fungi, insects, and nematodes. Until now, methyl iodide has not been evaluated against Bacillus anthracis. Sterne strain Bacillus anthracis spores were subjected to methyl iodide fumigation at room temperature and at 550C. Efficacy was measured on a log-scale with a 6-log reduction in CFUs being considered successful compared to the U.S. Environmental Protection Agency biocide standard. Such efficacies were obtained after just one hour at 55 °C and after 12 hours at room temperature. No detrimental effects were observed on glassware, PTFE O-rings, or stainless steel. This is the first reported efficacy of methyl iodide in the reduction of Bacillus anthracis spore contamination at ambient and elevated temperatures.

  8. Dendritic Cells Endocytose Bacillus Anthracis Spores: Implications for Anthrax Pathogenesis

    Science.gov (United States)

    2007-11-02

    Dendritic Cells Endocytose Bacillus anthracis Spores: Implications for Anthrax Pathogenesis1 Katherine C. Brittingham,* Gordon Ruthel,* Rekha G...germination and dissemination of spores. Found in high frequency throughout the respiratory track, dendritic cells (DCs) routinely take up foreign...COVERED - 4. TITLE AND SUBTITLE Dendritic cells endocytose Bacillus anthracis spores: implications for anthrax pathogenesis, The Journal of

  9. Architecture and Assembly of the Bacillus subtilis Spore Coat

    Science.gov (United States)

    2014-09-26

    icandy contaminated with germinated spores and these germinat ed spores were removed by centrifugation in a one step HistodenzTM (Sigma, St. Louis...spore resistance but also because some coat proteins play significant roles in spore germination . However, much recent work on the spore coat has... germinating spores of various Bacillus [14,21 30] and Clostridium [3 1] species. H owever, this analysis has generally been conducted on wild type

  10. FORMALDEHYDE GAS INACTIVATION OF BACILLUS ANTHRACIS, BACILLUS SUBTILIS AND GEOBACILLUS STEAROTHERMOPHILUS SPORES ON INDOOR SURFACE MATERIALS.

    Science.gov (United States)

    Research evaluated the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface material using formaldehyde gas. Spores were dried on seven types of indoor surfaces and exposed to 1100 ppm formaldehyde gas for 10 hr. Fo...

  11. Structural Analysis of Bacillus subtilis Spore Peptidoglycan During Sporulation

    OpenAIRE

    2000-01-01

    Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation:Jennifer L. Meador-Parton:David L. Popham, Chairman:Department of Biology:(ABSTRACT):Bacterial spore peptidoglycan (PG) is very loosely cross-linked relative to vegetative PG. Theories suggest that loosely cross-linked spore PG may have a flexibility which contributes to the attainment of spore core dehydration. The structure of the PG found in fully dormant spores has previously been examined in wild type and m...

  12. Nanomechanical Characterization of Bacillus anthracis Spores by Atomic Force Microscopy

    OpenAIRE

    2016-01-01

    The study of structures and properties of bacterial spores is important to understanding spore formation and biological responses to environmental stresses. While significant progress has been made over the years in elucidating the multilayer architecture of spores, the mechanical properties of the spore interior are not known. Here, we present a thermal atomic force microscopy (AFM) study of the nanomechanical properties of internal structures of Bacillus anthracis spores. We developed a nan...

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

    Science.gov (United States)

    2015-06-19

    2015): << Inhibiting inosine hydrolase and alanine racemase to enhance the germination of Bacillus anthracis Sterne spores: potential spore...inosine hydrolase and alanine racemase to enhance the germination of Bacillus anthracis Sterne spores potential spore decontamination strategies 5a...EASIER, SAFER, and CHEAPER Inducing spore germination should make resulting bacteria much more susceptible to decontamination methods and will be

  14. Inactivation of Bacillus Anthracis Spores Using Carbon Nanotubes

    Science.gov (United States)

    2014-10-30

    2010 31-May-2014 Approved for Public Release; Distribution Unlimited Final Report: (Life Science Division/Biochemistry) Inactivation of Bacillus ...S) AND ADDRESS (ES) U.S. Army Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 Bacillus Anthracis, Spores, Biofilm, Inhibition...Biochemistry) Inactivation of Bacillus Anthracis Spores Using Carbon Nanotubes Report Title The Specific Aims of the project were to investigate: 1) the

  15. INCORPORATION OF BACTERIOPHAGE GENOME BY SPORES OF BACILLUS SUBTILIS.

    Science.gov (United States)

    TAKAHASHI, I

    1964-06-01

    Takahashi, I. (Microbiology Research Institute, Ottawa, Ontario, Canada). Incorporation of bacteriophage genome by spores of Bacillus subtilis. J. Bacteriol. 87:1499-1502. 1964-The buoyant density in a CsCl gradient of deoxyribonucleic acid (DNA) extracted from spores of Bacillus subtilis was found to be identical to that of DNA from vegetative cells. Density-gradient centrifugation of DNA of spores derived from cultures infected with phage PBS 1 revealed the presence of a minor band whose density corresponded to that of the phage DNA in addition to the spore DNA. No intermediate bands were present. The relative amount of the phage DNA present in the spores was estimated to be 11%, suggesting that spores of this organism may incorporate several copies of the phage genome. Although the possibility that true lysogeny may occur cannot be entirely eliminated, the results seem to indicate that the phage genomes incorporated into spores are not attached to the host chromosome in this system.

  16. Comparison of hand hygiene procedures for removing Bacillus cereus spores.

    Science.gov (United States)

    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.

  17. Electron Beam Irradiation Dose Dependently Damages the Bacillus Spore Coat and Spore Membrane

    Directory of Open Access Journals (Sweden)

    S. E. Fiester

    2012-01-01

    Full Text Available Effective control of spore-forming bacilli begs suitable physical or chemical methods. While many spore inactivation techniques have been proven effective, electron beam (EB irradiation has been frequently chosen to eradicate Bacillus spores. Despite its widespread use, there are limited data evaluating the effects of EB irradiation on Bacillus spores. To study this, B. atrophaeus spores were purified, suspended in sterile, distilled water, and irradiated with EB (up to 20 kGy. Irradiated spores were found (1 to contain structural damage as observed by electron microscopy, (2 to have spilled cytoplasmic contents as measured by spectroscopy, (3 to have reduced membrane integrity as determined by fluorescence cytometry, and (4 to have fragmented genomic DNA as measured by gel electrophoresis, all in a dose-dependent manner. Additionally, cytometry data reveal decreased spore size, increased surface alterations, and increased uptake of propidium iodide, with increasing EB dose, suggesting spore coat alterations with membrane damage, prior to loss of spore viability. The present study suggests that EB irradiation of spores in water results in substantial structural damage of the spore coat and inner membrane, and that, along with DNA fragmentation, results in dose-dependent spore inactivation.

  18. Live-imaging of Bacillus subtilis spore germination and outgrowth

    NARCIS (Netherlands)

    Pandey, R.

    2014-01-01

    Spores of Gram-positive bacteria such as Bacillus and Clostridium cause huge economic losses to the food industry. In food products, spores survive under food preservation conditions and subsequent germination and outgrowth eventually causes food spoilage. Therefore efforts are being made to elimina

  19. Binding Affinity of Glycoconjugates to BACILLUS Spores and Toxins

    Science.gov (United States)

    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.

  20. Flow-cytometric Analysis of Bacillus anthracis Spores

    Directory of Open Access Journals (Sweden)

    D. V. Kamboj

    2006-11-01

    Full Text Available Flow-cytometric technique has been established as a powerful tool for detection andidentification of microbiological agents. Unambiguous and rapid detection of Bacillus anthracisspores has been reported using immunoglobulin G-fluorescein isothiocyanate conjugate againstlive spores. In addition to the high sensitivity, the present technique could differentiate betweenspores of closely related species, eg, Bacillus cereus and Bacillus subtilis using fluorescenceintensity. The technique can be used for detection of live as well as inactivated spores makingit more congenial for screening of suspected samples of bioterrorism.

  1. Bacillus atrophaeus Outer Spore Coat Assembly and Ultrastructure

    Energy Technology Data Exchange (ETDEWEB)

    Plomp, M; Leighton, T J; Wheeler, K E; Pitesky, M E; Malkin, A J

    2005-11-21

    Our previous atomic force microscopy (AFM) studies successfully visualized native Bacillus atrophaeus spore coat ultrastructure and surface morphology. We have shown that the outer spore coat surface is formed by a crystalline array of {approx}11 nm thick rodlets, having a periodicity of {approx}8 nm. We present here further AFM ultrastructural investigations of air-dried and fully hydrated spore surface architecture. In the rodlet layer, planar and point defects, as well as domain boundaries, similar to those described for inorganic and macromolecular crystals, were identified. For several Bacillus species, rodlet structure assembly and architectural variation appear to be a consequence of species-specific nucleation and crystallization mechanisms that regulate the formation of the outer spore coat. We propose a unifying mechanism for nucleation and self-assembly of this crystalline layer on the outer spore coat surface.

  2. DECONTAMINATION ASSESSMENT OF BACILLUS ANTHRACIS, BACILLUS SUBTILIS, AND GEOBACILLUS STEAROTHERMOPHILUS SPORES ON INDOOR SURFACTS USING A HYDROGEN PERIOXIDE GAS GENERATOR

    Science.gov (United States)

    Aims: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. Methods and Results: B. anthracis, B. subtilis, and G. Stearothermophilus spores were dried on seven...

  3. UV resistance of Bacillus anthracis spores revisited: validation of Bacillus subtilis spores as UV surrogates for spores of B. anthracis Sterne.

    Science.gov (United States)

    Nicholson, Wayne L; Galeano, Belinda

    2003-02-01

    Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.

  4. Presence survival spores of Bacillus thuringiensis varieties in grain warehouse

    Directory of Open Access Journals (Sweden)

    Sánchez-Yáñez Juan Manuel

    2016-08-01

    Full Text Available Genus Bacillus thuringiensis (Bt synthesized spores and crystals toxic to pest-insects in agriculture. Bt is comospolitan then possible to isolate some subspecies or varieties from warehouse. The aims of study were: i to isolate Bt varieties from grain at werehouse ii to evaluate Bt toxicity on Spodoptera frugiperda and Shit-ophilus zeamaisese iii to analyze Bt spores persistence in Zea mays grains at werehouse compared to same Bt on grains exposed to sun radiation. Results showed that at werehouse were recovered more than one variety of Bt spores. According to each isolate Bt1 o Bt2 were toxic to S. frugiperda or S. zeamaisese. One those Bt belong to var morrisoni. At werehouse these spores on Z. mays grains surviving more time, while the same spores exposed to boicide sun radiation they died.

  5. Quantitative and Sensitive RNA Based Detection of Bacillus Spores

    Directory of Open Access Journals (Sweden)

    Ekaterina eOsmekhina

    2014-03-01

    Full Text Available The fast and reliable detection of bacterial spores is of great importance and still remains a challenge. Here we describe a direct RNA based diagnostic method for the specific detection of viable bacterial spores which does not depends on an enzymatic amplification step and therefore is directly appropriate for quantification. The procedure includes the following steps: (i heat activation of spores, (ii germination and enrichment cultivation, (iii cell lysis, and (iv analysis of 16S rRNA in crude cell lysates using a sandwich hybridization assay. The sensitivity of the method is dependent on the cultivation time and the detection limit; it is possible to detect 10 spores per ml when the RNA analysis is performed after 6 h of enrichment cultivation. At spore concentrations above 106 spores per ml the cultivation time can be shortened to 30 min. Total analysis times are in the range of 2 to 8 hours depending on the spore concentration in samples. The developed procedure is optimized at the example of Bacillus subtilis spores but should be applicable to other organisms. The new method can easily be modified for other target RNAs and is suitable for specific detection of spores from known groups of organisms.

  6. Decontamination Options for Drinking Water Contaminated with Bacillus anthracis Spores

    Energy Technology Data Exchange (ETDEWEB)

    Raber, E; Burklund, A

    2010-02-16

    Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination options for use in a contaminated drinking water supply. The parameters were: (1) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus); (2) spore concentration in suspension (10{sup 2} to 10{sup 6} spores/ml); (3) chemical characteristics of decontaminant [sodium dicholor-s-triazinetrione dihydrate (Dichlor), hydrogen peroxide, potassium peroxymonosulfate (Oxone), sodium hypochlorite, and VirkonS{reg_sign}]; (4) decontaminant concentration (0.01% to 5%); and (5) decontaminant exposure time (10 min to 24 hr). Results from 162 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5%, and Dichlor and sodium hypochlorite at a concentration of 2%, were 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 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 EPA's biocide standard of greater than a 6 log kill after a 10-minute exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS{reg_sign} and Oxone were less effective decontaminants than other options evaluated in this study and did not meet the EPA's efficacy standard for biocides. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult.

  7. Architecture and High-Resolution Structure of Bacillus thuringiensis and Bacillus cereus Spore Coat Surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Plomp, M; Leighton, T; Wheeler, K; Malkin, A

    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. cereus 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.

  8. 14C Analysis of protein extracts from Bacillus spores.

    Science.gov (United States)

    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.

  9. An Optical Biosensor for Bacillus Cereus Spore Detection

    Science.gov (United States)

    Li, Chengquan; Tom, Harry W. K.

    2005-03-01

    We demonstrate a new transduction scheme for optical biosensing. Bacillus cereus is a pathogen that may be found in food and dairy products and is able to produce toxins and cause food poisoning. It is related to Bacillus anthracis (anthrax). A CCD array covered with micro-structured glass coverslip is used to detect the optical resonant shift due to the binding of the antigen (bacillus cereus spore) to the antibody (polyclonal antibody). This novel optical biosensor scheme has the potential for detecting 10˜100 bioagents in a single device as well as the potential to test for antigens with multiple antibody tests to avoid ``false positives.''

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

    OpenAIRE

    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 ...

  11. A method for the determination of bacterial spore DNA content based on isotopic labelling, spore germination and diphenylamine assay; ploidy of spores of several Bacillus species.

    Science.gov (United States)

    Hauser, P M; Karamata, D

    1992-01-01

    A reliable method for measuring the spore DNA content, based on radioactive DNA labelling, spore germination in absence of DNA replication and diphenylamine assay, was developed. The accuracy of the method, within 10-15%, is adequate for determining the number of chromosomes per spore, provided that the genome size is known. B subtilis spores were shown to be invariably monogenomic, while those of larger bacilli Bacillus megaterium, Bacillus cereus and Bacillus thuringiensis, often, if not invariably, contain two genomes. Attempts to modify the spore DNA content of B subtilis by altering the richness of the sporulation medium, the sporulation conditions (liquid or solid medium), or by mutation, were apparently unsuccessful. An increase of spore size with medium richness, not accompanied by an increase in DNA content, was observed. The implication of the apparently species-specific spore ploidy and the influence of the sporulation conditions on spore size and shape are discussed.

  12. Simple detection of Bacillus anthracis spores by precipitation method with goat antibody anti anthrosa

    OpenAIRE

    2016-01-01

    Background: Bacillus anthracis has a potential for biological weapon or bioterorism. Attack of Bacillus anthracis is very fatal, and the distribution is very easy and cheap through the spores. The aim of this was study to detect the spores of Bacillus anthracis. Methods: Bacillus anthracis isolates were grown on serum agar and then sheep blood medium, to stimulate capsule formation. Spores which formed painted using the method of Schaefer and Fultton. The methods of precipitation and immun...

  13. Multigeneration Cross-Contamination of Mail with Bacillus anthracis Spores.

    Directory of Open Access Journals (Sweden)

    Jason Edmonds

    Full Text Available The release of biological agents, including those which could be used in biowarfare or bioterrorism in large urban areas, has been a concern for governments for nearly three decades. Previous incidents from Sverdlosk and the postal anthrax attack of 2001 have raised questions on the mechanism of spread of Bacillus anthracis spores as an aerosol or contaminant. Prior studies have demonstrated that Bacillus atrophaeus is easily transferred through simulated mail handing, but no reports have demonstrated this ability with Bacillus anthracis spores, which have morphological differences that may affect adhesion properties between spore and formite. In this study, equipment developed to simulate interactions across three generations of envelopes subjected to tumbling and mixing was used to evaluate the potential for cross-contamination of B. anthracis spores in simulated mail handling. In these experiments, we found that the potential for cross-contamination through letter tumbling from one generation to the next varied between generations while the presence of a fluidizer had no statistical impact on the transfer of material. Likewise, the presence or absence of a fluidizer had no statistically significant impact on cross-contamination levels or reaerosolization from letter opening.

  14. Multigeneration Cross-Contamination of Mail with Bacillus anthracis Spores

    Science.gov (United States)

    Edmonds, Jason; Lindquist, H. D. Alan; Sabol, Jonathan; Martinez, Kenneth; Shadomy, Sean; Cymet, Tyler; Emanuel, Peter

    2016-01-01

    The release of biological agents, including those which could be used in biowarfare or bioterrorism in large urban areas, has been a concern for governments for nearly three decades. Previous incidents from Sverdlosk and the postal anthrax attack of 2001 have raised questions on the mechanism of spread of Bacillus anthracis spores as an aerosol or contaminant. Prior studies have demonstrated that Bacillus atrophaeus is easily transferred through simulated mail handing, but no reports have demonstrated this ability with Bacillus anthracis spores, which have morphological differences that may affect adhesion properties between spore and formite. In this study, equipment developed to simulate interactions across three generations of envelopes subjected to tumbling and mixing was used to evaluate the potential for cross-contamination of B. anthracis spores in simulated mail handling. In these experiments, we found that the potential for cross-contamination through letter tumbling from one generation to the next varied between generations while the presence of a fluidizer had no statistical impact on the transfer of material. Likewise, the presence or absence of a fluidizer had no statistically significant impact on cross-contamination levels or reaerosolization from letter opening. PMID:27123934

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

    Science.gov (United States)

    2014-01-01

    SECURITY CLASSIFICATION OF: The germination of multiple individual Bacillus subtilis spores by a high pressure (HP) of 140-150 (unless noted...otherwise) megaPascals (MPa) that activates spore germinant receptors (GRs) was monitored by phase contrast microscopy in a diamond anvil cell. Major...conclusions were that: i) >95% of spores germinated in 40 min; ii) individual spore’s HP germination kinetics were very similar to those for nutrient

  16. Studies on the bacterial spore coat 6 effects of alkali extraction on the spore of Bacillus thiaminolyticus.

    Science.gov (United States)

    Minami, J; Ichikawa, T; Kondo, M

    1977-01-01

    Thin sections of the spore of Bacillus thiaminolyticus Matsukawa and Misawa show a characteristic surface structure with five ridges, and a series of three district layers. The outer layer of the spore coat was peeled off by SDS sonic treatment, and than the middle layer was solubilized by alkali extraction of the SDS sonic-treated spore. The spores subjected to these treatments were still refractile, heat resistant, and contained dipicolinic acid, but lost their resistance to mechanical shock.

  17. High-Resolution Spore Coat Architecture and Assembly of Bacillus Spores

    Energy Technology Data Exchange (ETDEWEB)

    Malkin, A J; Elhadj, S; Plomp, M

    2011-03-14

    Elucidating the molecular architecture of bacterial and cellular surfaces and its structural dynamics is essential to understanding mechanisms of pathogenesis, immune response, physicochemical interactions, environmental resistance, and provide the means for identifying spore formulation and processing attributes. I will discuss the application of in vitro atomic force microscopy (AFM) for studies of high-resolution coat architecture and assembly of several Bacillus spore species. We have demonstrated that bacterial spore coat structures are phylogenetically and growth medium determined. We have proposed that strikingly different species-dependent coat structures of bacterial spore species are a consequence of sporulation media-dependent nucleation and crystallization mechanisms that regulate the assembly of the outer spore coat. Spore coat layers were found to exhibit screw dislocations and two-dimensional nuclei typically observed on inorganic and macromolecular crystals. This presents the first case of non-mineral crystal growth patterns being revealed for a biological organism, which provides an unexpected example of nature exploiting fundamental materials science mechanisms for the morphogenetic control of biological ultrastructures. We have discovered and validated, distinctive formulation-specific high-resolution structural spore coat and dimensional signatures of B. anthracis spores (Sterne strain) grown in different formulation condition. We further demonstrated that measurement of the dimensional characteristics of B. anthracis spores provides formulation classification and sample matching with high sensitivity and specificity. I will present data on the development of an AFM-based immunolabeling technique for the proteomic mapping of macromolecular structures on the B. anthracis surfaces. These studies demonstrate that AFM can probe microbial surface architecture, environmental dynamics and the life cycle of bacterial and cellular systems at near

  18. Challenges and advances in systems biology analysis of Bacillus spore physiology; molecular differences between an extreme heat resistant spore forming Bacillus subtilis food isolate and a laboratory strain

    NARCIS (Netherlands)

    Brul, S.; van Beilen, J.; Caspers, M.; O'Brien, A.; de Koster, C.; Oomes, S.; Smelt, J.; Kort, R.; ter Beek, A.

    2011-01-01

    Bacterial spore formers are prime organisms of concern in the food industry. Spores from the genus Bacillus are extremely stress resistant, most notably exemplified by high thermotolerance. This sometimes allows surviving spores to germinate and grow out to vegetative cells causing food spoilage and

  19. 14C Analysis of Protein Extracts from Bacillus Spores

    Science.gov (United States)

    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

  20. Vacuum-induced Mutations In Bacillus Subtilis Spores

    Science.gov (United States)

    Munakata, N.; Maeda, M.; Hieda, K.

    During irradiation experiments with vacuum-UV radiation using synchrotron sources, we made unexpected observation that Bacillus subtilis spores of several recombination-deficient strains lost colony-forming ability by the exposure to high vacuum alone. Since this suggested the possible injury in spore DNA, we looked for mutation induction using the spores of strains HA101 (wild-type repair capability) and TKJ6312 (excision and spore repair deficient) that did not lose survivability. It was found that the frequency of nalidixic-acid resistant mutation increased several times in both of these strains by the exposure to high vacuum (10e-4 Pa after 24 hours). The analysis of sequence changes in gyrA gene showed that the majority of mutations carried a unique allele (gyrA12) of tandem double-base substitutions from CA to TT. The observation has been extended to rifampicin resistant mutations, the majority of that carried substitutions from CA to TT or AT in rpoB gene. On the other hand, when the spores of strains PS578 and PS2319 (obtained from P. Setlow) that are defective in a group of small acidic proteins (alpha/beta-type SASP) were similarly treated, none of the mutants analyzed carried such changes. This suggests that the unique mutations might be induced by the interaction of small acidic proteins with spore DNA under forced dehydration. The results indicate that extreme vacuum causes severe damage in spore DNA, and provide additional constraint to the long-term survival of bacterial spores in the space environment.

  1. Achieving Consistent Multiple Daily Low-Dose Bacillus anthracis Spore Inhalation Exposures in the Rabbit Model

    Science.gov (United States)

    2012-06-13

    daily low-dose Bacillus anthracis spore inhalation exposures in the rabbit model Roy E. Barnewall 1, Jason E. Comer 1, Brian D. Miller 1, BradfordW...multiple exposure days. Keywords: Bacillus anthracis , inhalation exposures, low-dose, subchronic exposures, spores, anthrax, aerosol system INTRODUCTION... Bacillus Anthracis Spore Inhalation Exposures In The Rabbit Model 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d

  2. Inhibition of Bacillus cereus spore outgrowth and multiplication by chitosan.

    Science.gov (United States)

    Mellegård, Hilde; From, Cecilie; Christensen, Bjørn E; Granum, Per E

    2011-10-03

    Bacillus cereus is an endospore-forming bacterium able to cause food-associated illness. Different treatment processes are used in the food industry to reduce the number of spores and thereby the potential of foodborne disease. Chitosan is a polysaccharide with well-documented antibacterial activity towards vegetative cells. The activity against bacterial spores, spore germination and subsequent outgrowth and growth (the latter two events hereafter denoted (out)growth), however, is poorly documented. By using six different chitosans with defined macromolecular properties, we evaluated the effect of chitosan on Bacillus cereus spore germination and (out)growth using optical density assays and a dipicolinic acid release assay. (Out)growth was inhibited by chitosan, but germination was not. The action of chitosan was found to be concentration-dependent and also closely related to weight average molecular weight (M(w)) and fraction of acetylation (F(A)) of the biopolymer. Chitosans of low acetylation (F(A)=0.01 or 0.16) inhibited (out)growth more effectively than higher acetylated chitosans (F(A)=0.48). For the F(A)=0.16 chitosans with medium (56.8kDa) and higher M(w) (98.3kDa), a better (out)growth inhibition was observed compared to low M(w) (10.6kDa) chitosan. The same trend was not evident with chitosans of 0.48 acetylation, where the difference in activity between the low (19.6kDa) and high M(w) (163.0kDa) chitosans was only minor. In a spore test concentration corresponding to 10(2)-10(3)CFU/ml (spore numbers relevant to food), less chitosan was needed to suppress (out)growth compared to higher spore numbers (equivalent to 10(8)CFU/ml), as expected. No major differences in chitosan susceptibility between three different strains of B. cereus were detected. Our results contribute to a better understanding of chitosan activity towards bacterial spore germination and (out)growth.

  3. Mechanisms of Induction of Germination of Bacillus subtilis Spores by High Pressure

    OpenAIRE

    Paidhungat, Madan; Setlow, Barbara; Daniels, William B.; Hoover, Dallas; Papafragkou, Efstathia; Setlow, Peter

    2002-01-01

    Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not att...

  4. Adhesion of Spores of Bacillus thuringiensis on a Planar Surface

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Eunhyea [Georgia Institute of Technology; Kweon, Hyojin [Georgia Institute of Technology; Yiacoumi, Sotira [Georgia Institute of Technology; Lee, Ida [University of Tennessee, Knoxville (UTK); Joy, David Charles [ORNL; Palumbo, Anthony Vito [ORNL; Tsouris, Costas [ORNL

    2010-01-01

    Adhesion of spores of Bacillus thuringiensis (Bt) and spherical silica particles on surfaces was experimentally and theoretically investigated in this study. Topography analysis via atomic force microscopy (AFM) and electron microscopy indicates that Bt spores are rod shaped, {approx}1.3 {mu}m in length and {approx}0.8 {mu}m in diameter. The adhesion force of Bt spores and silica particles on gold-coated glass was measured at various relative humidity (RH) levels by AFM. It was expected that the adhesion force would vary with RH because the individual force components contributing to the adhesion force depend on RH. The adhesion force between a particle and a planar surface in atmospheric environments was modeled as the contribution of three major force components: capillary, van der Waals, and electrostatic interaction forces. Adhesion force measurements for Bt spore (silica particle) and the gold surface system were comparable with calculations. Modeling results show that there is a critical RH value, which depends on the hydrophobicity of the materials involved, below which the water meniscus does not form and the contribution of the capillary force is zero. As RH increases, the van der Waals force decreases while the capillary force increases to a maximum value.

  5. Sporicidal characteristics of heated dolomite powder against Bacillus subtilis spores.

    Science.gov (United States)

    Yasue, Syogo; Sawai, Jun; Kikuchi, Mikio; Nakakuki, Takahito; Sano, Kazuo; Kikuchi, Takahide

    2014-01-01

    Dolomite is a double salt composed of calcium carbonate (CaCO3) and magnesium carbonate (MgCO3). The heat treatment of CaCO3 and MgCO3 respectively generates calcium oxide (CaO) and magnesium oxide (MgO), which have antimicrobial activity. In this study, heated dolomite powder (HDP) slurry was investigated for its sporicidal activity against Bacillus subtilis ATCC 6633 spores. The B. subtilis spores used in this study were not affected by acidic (pH 1) or alkaline (pH 13) conditions, indicating that they were highly resistant. However, dolomite powder heated to 1000℃ for 1 h could kill B. subtilis spores, even at pH 12.7. Sporicidal activity was only apparent when the dolomite powder was heated to 800℃ or higher, and sporicidal activity increased with increases in the heating temperature. This temperature corresponded to that of the generation of CaO. We determined that MgO did not contribute to the sporicidal activity of HDP. To elucidate the sporicidal mechanism of the HDP against B. subtilis spores, the generation of active oxygen from HDP slurry was examined by chemiluminescence analysis. The generation of active oxygen increased when the HDP slurry concentration rose. The results suggested that, in addition to its alkalinity, the active oxygen species generated from HDP were associated with sporicidal activity.

  6. Setting risk-informed environmental standards for Bacillus anthracis spores.

    Science.gov (United States)

    Hong, Tao; Gurian, Patrick L; Ward, Nicholas F Dudley

    2010-10-01

    In many cases, human health risk from biological agents is associated with aerosol exposures. Because air concentrations decline rapidly after a release, it may be necessary to use concentrations found in other environmental media to infer future or past aerosol exposures. This article presents an approach for linking environmental concentrations of Bacillus. anthracis (B. anthracis) spores on walls, floors, ventilation system filters, and in human nasal passages with human health risk from exposure to B. anthracis spores. This approach is then used to calculate example values of risk-informed concentration standards for both retrospective risk mitigation (e.g., prophylactic antibiotics) and prospective risk mitigation (e.g., environmental clean up and reoccupancy). A large number of assumptions are required to calculate these values, and the resulting values have large uncertainties associated with them. The values calculated here suggest that documenting compliance with risks in the range of 10(-4) to 10(-6) would be challenging for small diameter (respirable) spore particles. For less stringent risk targets and for releases of larger diameter particles (which are less respirable and hence less hazardous), environmental sampling would be more promising.

  7. Mutagenesis of Bacillus subtilis spores exposed to simulated space environment

    Science.gov (United States)

    Munakata, N.; Natsume, T.; Takahashi, K.; Hieda, K.; Panitz, C.; Horneck, G.

    Bacterial spores can endure in a variety of extreme earthly environments. However, some conditions encountered during the space flight could be detrimental to DNA in the spore, delimiting the possibility of transpermia. We investigate the genetic consequences of the exposure to space environments in a series of preflight simulation project of EXPOSE. Using Bacillus subtilis spores of repair-proficient HA101 and repair-deficient TKJ6312 strains, the mutations conferring resistance to rifampicin were detected, isolated and sequenced. Most of the mutations were located in a N-terminal region of the rpoB gene encoding RNA polymerase beta-subunit. Among several potentially mutagenic factors, high vacuum, UV radiation, heat, and accelerated heavy ions induced mutations with varying efficiencies. A majority of mutations induced by vacuum exposure carried a tandem double-base change (CA to TT) at a unique sequence context of TCAGC. Results indicate that the vacuum and high temperature may act synergistically for the induction of mutations.

  8. Effects of Mn2+ Levels on the Resistance Properties of Bacillus cereus Spores

    Science.gov (United States)

    2013-01-01

    In contrast, Bacillus subtilis spores with over a 200-fold range of protoplast Mn levels exhibited no significant differences in resistance to...Bacillus megaterium by wet heat. Lett. Appl . Microbiol. 50:507-514. Daly MJ (2012) Death by protein damage in irradiated cells. DNA Repair 11:12-21...levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide. J. Appl . Microbiol. 111:663-670. Ghosh S, Setlow P (2010

  9. NanoSIMS analysis of Bacillus spores for forensics

    Energy Technology Data Exchange (ETDEWEB)

    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 of 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

  10. Observations on the migration of bacillus spores outside a contaminated facility during a decontamination efficacy study

    Science.gov (United States)

    Silvestri, Erin E.; Perkins, Sarah; Lordo, Robert; Kovacik, William; Nichols, Tonya L.; Bowling, Charlena Yoder; Griffin, Dale W.; Schaefer, Frank W.

    2015-01-01

    The potential for an intentional wide-area or indoor release of Bacillus anthracis spores remains a concern, but the fate and transport of B. anthracis spores in indoor and outdoor environments are not well understood. Some studies have examined the possibility of spore transport within ventilation systems and in buildings and transport into a building following an outdoor release. Little research exists regarding the potential for spores to migrate to the outside of a building following an indoor release.

  11. Impact of Spore Biology on the Rate of Kill and Suppression of Resistance in Bacillus anthracis▿

    OpenAIRE

    Drusano, G L; Okusanya, O. O.; Okusanya, A. O.; van Scoy, B.; Brown, D L; Fregeau, C.; Kulawy, R.; Kinzig, M; Sörgel, F; Heine, H. S.; Louie, A

    2009-01-01

    Bacillus anthracis is complex because of its spore form. The spore is invulnerable to antibiotic action. It also has an impact on the emergence of resistance. We employed the hollow-fiber infection model to study the impacts of different doses and schedules of moxifloxacin on the total-organism population, the spore population, and the subpopulations of vegetative- and spore-phase organisms that were resistant to moxifloxacin. We then generated a mathematical model of the impact of moxifloxac...

  12. Evaluating Composite Sampling Methods of Bacillus spores at Low Concentrations

    Energy Technology Data Exchange (ETDEWEB)

    Hess, Becky M.; Amidan, Brett G.; Anderson, Kevin K.; Hutchison, Janine R.

    2016-10-13

    Restoring facility operations after the 2001 Amerithrax attacks took over three months to complete, highlighting the need to reduce remediation time. The most time intensive tasks were environmental sampling and sample analyses. Composite sampling allows disparate samples to be combined, with only a single analysis needed, making it a promising method to reduce response times. We developed a statistical experimental design to test three different composite sampling methods: 1) single medium single pass composite: a single cellulose sponge samples multiple coupons; 2) single medium multi-pass composite: a single cellulose sponge is used to sample multiple coupons; and 3) multi-medium post-sample composite: a single cellulose sponge samples a single surface, and then multiple sponges are combined during sample extraction. Five spore concentrations of Bacillus atrophaeus Nakamura spores were tested; concentrations ranged from 5 to 100 CFU/coupon (0.00775 to 0.155CFU/cm2, respectively). Study variables included four clean surface materials (stainless steel, vinyl tile, ceramic tile, and painted wallboard) and three grime coated/dirty materials (stainless steel, vinyl tile, and ceramic tile). Analysis of variance for the clean study showed two significant factors: composite method (p-value < 0.0001) and coupon material (p-value = 0.0008). Recovery efficiency (RE) was higher overall using the post-sample composite (PSC) method compared to single medium composite from both clean and grime coated materials. RE with the PSC method for concentrations tested (10 to 100 CFU/coupon) was similar for ceramic tile, painted wall board, and stainless steel for clean materials. RE was lowest for vinyl tile with both composite methods. Statistical tests for the dirty study showed RE was significantly higher for vinyl and stainless steel materials, but significantly lower for ceramic tile. These results suggest post-sample compositing can be used to reduce sample analysis time when

  13. High Pressure Germination of Bacillus subtilis Spores with Alterations in Levels and Types of Germination Proteins

    Science.gov (United States)

    2014-01-01

    1ITLE AND SUBTITLE 5a CONTRACTNUMBER High pressure germination of Bacillus subtilis spores with W911NF-09-l-0286 alterations in levels and types of...A moderate high pressure (mHP) of 150 megaPascals (MPa) triggers germination of Bacillus subtilis spores via germinant receptors (GRs), while...germination by a very high pressure (vHP) of550 MPa is GR-independent. The mHP and vHP germination of Bacillus subtilis spores with different levels ofGRs

  14. Inactivation and ultrastructure analysis of Bacillus spp. and Clostridium perfringens spores.

    Science.gov (United States)

    Brantner, Christine A; Hannah, Ryan M; Burans, James P; Pope, Robert K

    2014-02-01

    Bacterial endospores are resistant to many environmental factors from temperature extremes to ultraviolet irradiation and are generally more difficult to inactivate or kill than vegetative bacterial cells. It is often considered necessary to treat spores or samples containing spores with chemical fixative solutions for prolonged periods of time (e.g., 1-21 days) to achieve fixation/inactivation to enable electron microscopy (EM) examination outside of containment laboratories. Prolonged exposure to chemical fixatives, however, can alter the ultrastructure of spores for EM analyses. This study was undertaken to determine the minimum amount of time required to inactivate/sterilize and fix spore preparations from several bacterial species using a universal fixative solution for EM that maintains the ultrastructural integrity of the spores. We show that a solution of 4% paraformaldehyde with 1% glutaraldehyde inactivated spore preparations of Bacillus anthracis, Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis, and Clostridium perfringens in 30 min, and Bacillus subtilis in 240 min. These results suggest that this fixative solution can be used to inactivate and fix spores from several major groups of bacterial spore formers after 240 min, enabling the fixed preparations to be removed from biocontainment and safely analyzed by EM outside of biocontainment.

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

    Science.gov (United States)

    2006-01-01

    in some strains of Bacillus cereus and Bacillus thuringiensis [55, 56]. The Ba813 marker has been used for a real time PCR assay using Taqman-type...pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes, J. Bacteriol. 181, 6509-6515 (1999). [36] L.B. Price, M. Hugh-Jones, P. J...useful results. The spores of Bacillus anthracis (BA) are particular- ly dangerous because they persist in the environment, and relatively small numbers

  16. The Adsorption Properties of Bacillus atrophaeus Spore on Functionalized Carbon Nanotubes

    Directory of Open Access Journals (Sweden)

    P. Cortes

    2010-01-01

    Full Text Available An equilibrium study of Bacillus atrophaeus (B.a spores on functionalized Single-Wall Carbon Nanotubes (SWCNTs has been performed in order to characterize the adsorption properties of the spores/nanotubes complex. The carbon nanotubes here investigated were subjected to a two-step purification and functionalization treatment in order to introduce chemical groups on their basal planes. The inclusion of carboxyl functional groups on the nanotubes was corroborated by Raman and infrared spectroscopy. These carboxyl groups appear to enhance the nanotube-B.a. interaction by reacting with the proteinaceous pili appendages present on the spore surface. The adsorption data demonstrate that bacillus spores diffuse faster on functionalized carbon nanotubes than on as-received and purified nanomaterials. Transmission Electron Microscopy also shows that the chemically treated nanotubes resulted in a swollen nano-network which seems to further enhance the bacillus adsorption due to a more extensive spore-nanotube contact area.

  17. Effects of Electrolyzed Oxidizing Water on Inactivation of Bacillus subtilis and Bacillus cereus Spores in Suspension and on Carriers.

    Science.gov (United States)

    Zhang, Chunling; Li, Baoming; Jadeja, Ravirajsinh; Hung, Yen-Con

    2016-01-01

    Spores of some Bacillus species are responsible for food spoilage and foodborne disease. These spores are highly resistant to various interventions and cooking processes. In this study, the sporicidal efficacy of acidic electrolyzed oxidizing (EO) water (AEW) and slightly acidic EO water (SAEW) with available chlorine concentration (ACC) of 40, 60, 80, 100, and 120 mg/L and treatment time for 1, 2, 3, 4, 5, and 6 min were tested on Bacillus subtilis and Bacillus cereus spores in suspension and on carrier with or without organics. The reduction of spore significantly increased with increasing ACC and treatment time (P waters containing 120 mg/L ACC, while only SAEW at 120 mg/L and 2 min treatment achieved >6 log reductions of B. subtilis spore. Both types of EO water with ACC of 60 mg/L and 6 min treatment achieved a reduction of B. subtilis and B. cereus spores to nondetectable level. EO water with ACC of 80 mg/L and treatment time of 3 min on carrier test without organics addition resulted in reductions of B. subtilis spore to nondetectable level. But, addition of 0.3% organics on carrier decreased the inactivation effect of EO water. This study indicated that EO water was highly effective in inactivation of B. subtilis and B. cereus spores in suspension or on carrier, and therefore, rendered it as a promising disinfectant to be applied in food industry.

  18. Dynamic localization of penicillin-binding proteins during spore development in Bacillus subtilis

    NARCIS (Netherlands)

    Scheffers, Dirk-Jan

    2005-01-01

    During Bacillus subtilis spore formation, many membrane proteins that function in spore development localize to the prespore septum and, subsequently, to the outer prespore membrane. Recently, it was shown that the cell-division-specific penicillin-binding proteins (PBPs) 1 and 2b localize to the as

  19. Bacillus subtilis spore protein SpoVAC functions as a mechanosensitive channel

    NARCIS (Netherlands)

    Velasquez Guzman, Jeanette; Schuurman-Wolters, Geesina; Birkner, Jan Peter; Abee, Tjakko; Poolman, Bert

    2014-01-01

    A critical event during spore germination is the release of Ca-DPA (calcium in complex with dipicolinic acid). The mechanism of release of Ca-DPA through the inner membrane of the spore is not clear, but proteins encoded by the Bacillus subtilis spoVA operon are involved in the process. We cloned an

  20. Removal of Bacillus anthracis sterne spore from commercial unpasteurized liquid egg white using crossflow microfiltration

    Science.gov (United States)

    Current pasteurization technology used by the egg industry is ineffective for destruction of spores such as those of Bacillus anthracis (BA). The validity of a cross-flow microfiltration (MF) process for separation of the attenuated strain of BA (Sterne) spores from commercial unpasteurized liquid ...

  1. Gas discharge plasmas are effective in inactivating Bacillus and Clostridium spores.

    Science.gov (United States)

    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.

  2. Regulation of the Spore Cortex Lytic Enzyme SleB in Bacillus anthracis

    OpenAIRE

    2014-01-01

    Bacillus anthracis is the causative agent of the disease anthrax and poses a threat due to its potential to be used as a biological weapon. The spore form of this bacterium is an extremely resistant structure, making spore decontamination exceptionally challenging. During spore germination, nutrient germinants interact with Ger receptors, triggering a cascade of events. A crucial event in this process is degradation of the cortex peptidoglycan by germination-specific lytic enzymes (GSLEs),...

  3. Germination of Spores of Astrobiologically Relevant Bacillus Species in High-Salinity Environments

    Science.gov (United States)

    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.

  4. Germinant-enhanced decontamination of Bacillus spores adhered to iron and cement-mortar drinking water infrastructures.

    Science.gov (United States)

    Szabo, Jeffrey G; Muhammad, Nur; Heckman, Lee; Rice, Eugene W; Hall, John

    2012-04-01

    Germination was evaluated as an enhancement to decontamination methods for removing Bacillus spores from drinking water infrastructure. Germinating spores before chlorinating cement mortar or flushing corroded iron was more effective than chlorinating or flushing alone.

  5. Impact of spore biology on the rate of kill and suppression of resistance in Bacillus anthracis.

    Science.gov (United States)

    Drusano, G L; Okusanya, O O; Okusanya, A O; van Scoy, B; Brown, D L; Fregeau, C; Kulawy, R; Kinzig, M; Sörgel, F; Heine, H S; Louie, A

    2009-11-01

    Bacillus anthracis is complex because of its spore form. The spore is invulnerable to antibiotic action. It also has an impact on the emergence of resistance. We employed the hollow-fiber infection model to study the impacts of different doses and schedules of moxifloxacin on the total-organism population, the spore population, and the subpopulations of vegetative- and spore-phase organisms that were resistant to moxifloxacin. We then generated a mathematical model of the impact of moxifloxacin, administered by continuous infusion or once daily, on vegetative- and spore-phase organisms. The ratio of the rate constant for vegetative-phase cells going to spore phase (K(vs)) to the rate constant for spore-phase cells going to vegetative phase (K(sv)) determines the rate of organism clearance. The continuous-infusion drug profile is more easily sensed as a threat; the K(vs)/K(sv) ratio increases at lower drug exposures (possibly related to quorum sensing). This movement to spore phase protects the organism but makes the emergence of resistance less likely. Suppression of resistance requires a higher level of drug exposure with once-daily administration than with a continuous infusion, a difference that is related to vegetative-to-spore (and back) transitioning. Spore biology has a major impact on drug therapy and resistance suppression. These findings explain why all drugs of different classes have approximately the same rate of organism clearance for Bacillus anthracis.

  6. Role of dipicolinic acid in the germination, stability, and viability of spores of Bacillus subtilis.

    Science.gov (United States)

    Magge, Anil; Granger, Amanda C; Wahome, Paul G; Setlow, Barbara; Vepachedu, Venkata R; Loshon, Charles A; Peng, Lixin; Chen, De; Li, Yong-Qing; Setlow, Peter

    2008-07-01

    Spores of Bacillus subtilis spoVF strains that cannot synthesize dipicolinic acid (DPA) but take it up during sporulation were prepared in medium with various DPA concentrations, and the germination and viability of these spores as well as the DPA content in individual spores were measured. Levels of some other small molecules in DPA-less spores were also measured. These studies have allowed the following conclusions. (i) Spores with no DPA or low DPA levels that lack either the cortex-lytic enzyme (CLE) SleB or the receptors that respond to nutrient germinants could be isolated but were unstable and spontaneously initiated early steps in spore germination. (ii) Spores that lacked SleB and nutrient germinant receptors and also had low DPA levels were more stable. (iii) Spontaneous germination of spores with no DPA or low DPA levels was at least in part via activation of SleB. (iv) The other redundant CLE, CwlJ, was activated only by the release of high levels of DPA from spores. (v) Low levels of DPA were sufficient for the viability of spores that lacked most alpha/beta-type small, acid-soluble spore proteins. (vi) DPA levels accumulated in spores prepared in low-DPA-containing media varied greatly between individual spores, in contrast to the presence of more homogeneous DPA levels in individual spores made in media with high DPA concentrations. (vii) At least the great majority of spores of several spoVF strains that contained no DPA also lacked other major spore small molecules and had gone through some of the early reactions in spore germination.

  7. Nanoscale structural and mechanical analysis of Bacillus anthracis spores inactivated with rapid dry heating.

    Science.gov (United States)

    Xing, Yun; Li, Alex; Felker, Daniel L; Burggraf, Larry W

    2014-03-01

    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.

  8. Modeling Radiation Effectiveness for Inactivation of Bacillus Spores

    Science.gov (United States)

    2015-09-17

    will undergo germination which is the first step in the process by which bacteria transforms from a dormant spore into a vegetative cell [28]. The...order to survive a dose of radiation, a spore must repair its damaged DNA during germination . The DNA repair process is dependent on reactions catalyzed...in the next section. 2.2 Life Cycle of a Bacterial Spore A dormant spore is formed via a multi- step process called sporulation (refer to Figure 2.5

  9. Current Physical and SDS Extraction Methods Do Not Efficiently Remove Exosporium Proteins from Bacillus anthracis spores

    Science.gov (United States)

    Thompson, Brian M.; Binkley, Jana M.; Stewart, George C.

    2011-01-01

    Biochemical studies of the outermost spore layers of the Bacillus cereus family are hindered by difficulties in efficient dispersal of the external spore layers and difficulties in dissociating protein complexes that comprise the exosporium layer. Detergent and physical methods have been utilized to disrupt the exosporium layer. Herein we compare commonly used SDS extraction buffers used to extract spore proteins and demonstrate the incomplete extractability of the exosporium layer by these methods. Sonication and bead beating methods for exosporium layer removal were also examined. A combination of genetic and physical methods is the most effective for isolating proteins found in the spore exosporium. PMID:21338631

  10. Role of Spore Coat Proteins in the Resistance of Bacillus subtilis Spores to Caenorhabditis elegans Predation▿

    OpenAIRE

    2008-01-01

    Bacterial spores are resistant to a wide range of chemical and physical insults that are normally lethal for the vegetative form of the bacterium. While the integrity of the protein coat of the spore is crucial for spore survival in vitro, far less is known about how the coat provides protection in vivo against predation by ecologically relevant hosts. In particular, assays had characterized the in vitro resistance of spores to peptidoglycan-hydrolyzing enzymes like lysozyme that are also imp...

  11. Anthrax Toxins in Context of Bacillus anthracis Spores and Spore Germination.

    Science.gov (United States)

    Cote, Christopher K; Welkos, Susan L

    2015-08-17

    The interaction of anthrax toxin or toxin components with B. anthracis spores has been demonstrated. Germinating spores can produce significant amounts of toxin components very soon after the initiation of germination. In this review, we will summarize the work performed that has led to our understanding of toxin and spore interactions and discuss the complexities associated with these interactions.

  12. In vitro and in vivo analyses of the Bacillus anthracis spore cortex lytic protein SleL

    OpenAIRE

    2012-01-01

    The bacterial endospore is the most resilient biological structure known. Multiple protective integument layers shield the spore core and promote spore dehydration and dormancy. Dormancy is broken when a spore germinates and becomes a metabolically active vegetative cell. Germination requires the breakdown of a modified layer of peptidoglycan (PG) known as the spore cortex. This study reports in vitro and in vivo analyses of the Bacillus anthracis SleL protein. SleL is a spore cortex lytic en...

  13. Responses of Bacillus subtilis spores to space environment: results from experiments in space.

    Science.gov (United States)

    Horneck, G

    1993-02-01

    Onboard of several spacecrafts (Apollo 16, Spacelab 1, LDEF), spores of Bacillus subtilis were exposed to selected parameters of space, such as space vacuum, different spectral ranges of solar UV-radiation and cosmic rays, applied separately or in combination, and we have studied their survival and genetic changes after retrieval. The spores survive extended periods of time in space--up to several years--, if protected against the high influx of solar UV-radiation. Water desorption caused by the space vacuum leads to structural changes of the DNA; the consequences are an increased mutation frequency and altered photobiological properties of the spores. UV-effects, such as killing and mutagenesis, are augmented, if the spores are in space vacuum during irradiation. Vacuum-specific photoproducts which are different from the 'spore photoproduct' may cause the synergistic response of spores to the simultaneous action of UV and vacuum. The experiments provide an experimental test of certain steps of the panspermia hypothesis.

  14. Evaluation of surface sampling method performance for Bacillus Spores on clean and dirty outdoor surfaces.

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Mollye C.; Einfeld, Wayne; Boucher, Raymond M.; Brown, Gary Stephen; Tezak, Matthew Stephen

    2011-06-01

    Recovery of Bacillus atrophaeous spores from grime-treated and clean surfaces was measured in a controlled chamber study to assess sampling method performance. Outdoor surfaces investigated by wipe and vacuum sampling methods included stainless steel, glass, marble and concrete. Bacillus atrophaeous spores were used as a surrogate for Bacillus anthracis spores in this study designed to assess whether grime-coated surfaces significantly affected surface sampling method performance when compared to clean surfaces. A series of chamber tests were carried out in which known amounts of spores were allowed to gravitationally settle onto both clean and dirty surfaces. Reference coupons were co-located with test coupons in all chamber experiments to provide a quantitative measure of initial surface concentrations of spores on all surfaces, thereby allowing sampling recovery calculations. Results from these tests, carried out under both low and high humidity conditions, show that spore recovery from grime-coated surfaces is the same as or better than spore recovery from clean surfaces. Statistically significant differences between method performance for grime-coated and clean surfaces were observed in only about half of the chamber tests conducted.

  15. Challenges and advances in systems biology analysis of Bacillus spore physiology; molecular differences between an extreme heat resistant spore forming Bacillus subtilis food isolate and a laboratory strain.

    Science.gov (United States)

    Brul, Stanley; van Beilen, Johan; Caspers, Martien; O'Brien, Andrea; de Koster, Chris; Oomes, Suus; Smelt, Jan; Kort, Remco; Ter Beek, Alex

    2011-04-01

    Bacterial spore formers are prime organisms of concern in the food industry. Spores from the genus Bacillus are extremely stress resistant, most notably exemplified by high thermotolerance. This sometimes allows surviving spores to germinate and grow out to vegetative cells causing food spoilage and possible intoxication. Similar issues though more pending toward spore toxigenicity are observed for the anaerobic Clostridia. The paper indicates the nature of stress resistance and highlights contemporary molecular approaches to analyze the mechanistic basis of it in Bacilli. A molecular comparison between a laboratory strain and a food borne isolate, very similar at the genomic level to the laboratory strain but generating extremely heat resistant spores, is discussed. The approaches cover genome-wide genotyping, proteomics and genome-wide expression analyses studies. The analyses aim at gathering sufficient molecular information to be able to put together an initial framework for dynamic modelling of spore germination and outgrowth behaviour. Such emerging models should be developed both at the population and at the single spore level. Tools and challenges in achieving the latter are succinctly discussed.

  16. The role of a purine-specific nucleoside hydrolase in spore germination of Bacillus thuringiensis.

    Science.gov (United States)

    Liang, Liang; He, Xihong; Liu, Gang; Tan, Huarong

    2008-05-01

    A homologous gene (iunH) of a putative nucleoside hydrolase (NH), which had been identified from the exosporia of Bacillus cereus and Bacillus anthracis spores, was cloned from Bacillus thuringiensis subsp. kurstaki. Disruption of iunH did not affect the vegetative growth and sporulation of Bacillus thuringiensis, but promoted both inosine- and adenosine-induced spore germination. The inosine- or adenosine-induced germination rate decreased when the wild-type iunH gene was overexpressed in Bacillus thuringiensis. The iunH gene product was characterized as a purine-specific NH. The kinetic parameters of IunH with inosine as substrate were K(m)=399+/-115 microM, k(cat)=48.9+/-8.5 s(-1) and k(cat)/K(m)=1.23 x 10(5) M(-1) s(-1). The optimal pH and temperature for IunH were found to be pH 6 and 80 degrees C. Meanwhile, the specific activity of inosine hydrolase in intact spores of the wild-type strain with inosine as substrate was 2.89+/-0.23x10(-2) micromol min(-1) (mg dry wt)(-1). These results indicate that IunH is important in moderating inosine- or adenosine-induced germination of Bacillus thuringiensis spores.

  17. Association and decontamination of Bacillus spores in a simulated drinking water system.

    Science.gov (United States)

    Morrow, J B; Almeida, J L; Fitzgerald, L A; Cole, K D

    2008-12-01

    The objective of this work was to elucidate the disinfectant susceptibility of Bacillus anthracis Sterne (BA) and a commercial preparation of Bacillus thuringiensis (BT) spores associated with a simulated drinking water system. Biofilms composed of indigenous water system bacteria were accumulated on copper and polyvinyl chloride (PVC) pipe material surfaces in a low-flow pipe loop and uniformly mixed tank reactor (CDC biofilm reactor). Application of a distributed shear during spore contact resulted in approximately a 1.0 and 1.6 log10 increase in the number of spores associated with copper and PVC surfaces, respectively. Decontamination of spores in both free suspension and after association with biofilm-conditioned pipe materials was attempted using free chlorine and monochloramine. Associated spores required 5- to 10-fold higher disinfectant concentrations to observe the same reduction of viable spores as in suspension. High disinfectant concentrations (103 mg/L free chlorine and 49 mg/L monochloramine) yielded less than a 2-log10 reduction in viable associated spores after 60 min. Spores associated with biofilms on copper surfaces consistently yielded higher Ct values than PVC.

  18. Bacillus spore classification via surface-enhanced Raman spectroscopy and principal component analysis.

    Science.gov (United States)

    Guicheteau, J; Argue, L; Emge, D; Hyre, A; Jacobson, M; Christesen, S

    2008-03-01

    Surface-enhanced Raman spectroscopy (SERS) can provide rapid fingerprinting of biomaterial in a nondestructive manner. The adsorption of colloidal silver to biological material suppresses native biofluorescence while providing electromagnetic surface enhancement of the normal Raman signal. This work validates the applicability of qualitative SER spectroscopy for analysis of bacterial species by utilizing principal component analysis (PCA) to show discrimination of biological threat simulants, based upon multivariate statistical confidence limits bounding known data clusters. Gram-positive Bacillus spores (Bacillus atrophaeus, Bacillus anthracis, and Bacillus thuringiensis) are investigated along with the Gram-negative bacterium Pantoea agglomerans.

  19. Function of the SpoVAEa and SpoVAF Proteins of Bacillus subtilis Spores

    Science.gov (United States)

    2014-06-01

    1ITLE AND SUBTITLE 5a CONTRACTNUMBER Function of the SpoVAEa and SpoVAF proteins of Bacillus W911NF-09-1-0286 subtilis spores 5b. GRANT NUMBER 5c...ABSTRACT The Bacillus subtilis spoVAEa and spoVAF genes are expressed in developng spores as members of the spoVA operon that encodes proteins essential...8217\\ ;~ 1~~~4-~,.1. A\\ C’~~1T 1\\ D~ ~~,.1 C’~~1T 1\\ T’\\ ~-~ ,.1;~~1. •• 4-~,.1 ~:-:1~-1 •• ;~ ~~~~~~~ ~f:’ 15. SUBJECT TERMS Bacillus , spores SpoVA

  20. Decontamination options for Bacillus anthracis-contaminated drinking water determined from spore surrogate studies.

    Science.gov (United States)

    Raber, Ellen; Burklund, Alison

    2010-10-01

    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 (10(2) and 10(6) 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 10(2) spores/ml. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult.

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

    Science.gov (United States)

    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.

  2. Ultraviolet irradiation of DNA complexed with. alpha. /. beta. -type small, acid-soluble proteins from spores of Bacillus or Clostridium species makes spore photoproduct but not thymine dimers

    Energy Technology Data Exchange (ETDEWEB)

    Nicholson, W.L.; Setlow, B.; Setlow, P. (Univ. of Connecticut Health Center, Farmington (United States))

    1991-10-01

    UV irradiation of complexes of DNA and an {alpha}/{beta}-type small, acid-soluble protein (SASP) from Bacillus subtilis spores gave decreasing amounts of pyrimidine dimers and increasing amounts of spore photoproduct as the SASP/DNA ratio was increased. The yields of pyrimidine dimers and spore photoproduct were < 0.2% and 8% of total thymine, respectively, when DNA saturated with SASP was irradiated at 254 nm with 30 kJ/m{sup 2}; in the absence of SASP the yields were reversed - 4.5% and 0.3%, respectively. Complexes of DNA with {alpha}/{beta}-type SASP from Bacillus cereus, Bacillus megaterium, or Clostridium bifermentans spores also gave spore photoproduct upon UV irradiation. However, incubation of these SASPs with DNA under conditions preventing complex formation or use of mutant SASPs that do not form complexes did not affect the photoproducts formed in vitro. These results suggest that the UV photochemistry of bacterial spore DNA in vivo is due to the binding of {alpha}/{beta}-type SASP, a binding that is known to cause a change in DNA conformation in vitro from the B form to the A form. The yields of spore photoproduct in vitro were significantly lower than in vivo, perhaps because of the presence of substances other than SASP in spores. It is suggested that as these factors diffuse out in the first minutes of spore germination, spore photoproduct yields become similar to those observed for irradiation of SASP/DNA complexes in vitro.

  3. The Adsorption Properties of Bacillus atrophaeus Spores on Single-Wall Carbon Nanotubes

    Directory of Open Access Journals (Sweden)

    P. Cortes

    2009-01-01

    Full Text Available An adsorption equilibrium and a kinetic study of Bacillus atrophaeus on Single-Wall Carbon Nanotubes (SWCNTs were here performed to provide the basis for developing biosensor devices for detecting threatening micro-organisms in water supply systems. B. atrophaeus spores and carbon nanotubes were subjected to a batch adsorption process to document their equilibria and kinetics. Here, commercial nanotubes were either studied as received or were acid-purified before adsorption experiments. The Bacillus spores appear to show higher affinity towards the purified nanotubes than to the as-received nanomaterial. The effective diffusivity of the spores onto the purified nanotubes was found to be approximately 30 percent higher than onto the as-received nanotubes. It seems that the removal of amorphous carbon from the as-received nanotubes through a purification process yielded an intimate nantoubes-spore interaction as revealed by transmission electron microscopy. Freundlich model successfully correlated the adsorption equilibrium data for the nanotubes-spore interaction. Transmission electron micrographs showed extensive contact between the Bacillus and the purified nanotubes, but the association appeared less intimate between the spores and the as-received nanotubes.

  4. Role of YpeB in cortex hydrolysis during germination of Bacillus anthracis spores.

    Science.gov (United States)

    Bernhards, Casey B; Popham, David L

    2014-10-01

    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.

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

    Science.gov (United States)

    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

  6. Quantification of the impact of single and multiple mild stresses on outgrowth heterogeneity of Bacillus cereus spores

    NARCIS (Netherlands)

    Melis, van C.C.J.; Besten, den H.M.W.; Nierop Groot, M.N.; Abee, T.

    2014-01-01

    Outgrowth heterogeneity of bacterial spore populations complicates both prediction and efficient control of spore outgrowth. In this study, the impact of mild preservation stresses on outgrowth of Bacillus cereus ATCC 14579 spores was quantified during the first stages of outgrowth. Heterogeneity in

  7. Friction and Adhesion Forces of Bacillus thuringiensis Spores on Planar Surfaces in Atmospheric Systems

    Energy Technology Data Exchange (ETDEWEB)

    Kweon, Hyojin [Georgia Institute of Technology; Yiacoumi, Sotira [Georgia Institute of Technology; Tsouris, Costas [ORNL

    2011-01-01

    The kinetic friction force and the adhesion force of Bacillus thuringiensis spores on planar surfaces in atmospheric systems were studied using atomic force microscopy. The influence of relative humidity (RH) on these forces varied for different surface properties including hydrophobicity, roughness, and surface charge. The friction force of the spore was greater on a rougher surface than on mica, which is atomically flat. As RH increases, the friction force of the spores decreases on mica whereas it increases on rough surfaces. The influence of RH on the interaction forces between hydrophobic surfaces is not as strong as for hydrophilic surfaces. The friction force of the spore is linear to the sum of the adhesion force and normal load on the hydrophobic surface. The poorly defined surface structure of the spore and the adsorption of contaminants from the surrounding atmosphere are believed to cause a discrepancy between the calculated and measured adhesion forces.

  8. Control of Bacillus licheniformis spores isolated from dairy materials in yogurt production.

    Science.gov (United States)

    Tanaka, Takashi; Ito, Akiko; Kamikado, Hideaki

    2012-01-01

    To determine the effects of sporulation temperature and period on Bacillus licheniformis spore heat resistance, B. licheniformis strain No.25 spores were sporulated at 30, 37, 42, or 50°C for 11 d and at 50°C for 1.7, 4, 7, or 11 d. The heat resistance of B. licheniformis strain No.25 spores at 110°C increased with an increase in the sporulation temperature. Spores sporulated at 50°C were 1.4-fold more heat resistant than those sporulated at 30°C. Furthermore, the heat resistance of B. licheniformis strain No.25 spores at 110°C increased with an increase in the sporulation period. Spores sporulated for 11 d were 5.3-fold more heat resistant than those sporulated for 1.7 d. The heat resistance of B. licheniformis strain No.25 spores at 110°C increased with increases in the sporulation temperature and sporulation period. The results presented in this study can be applied to the pasteurization process to control B. licheniformis spores. Pasteurization at 110°C for about 60sec. is effective in controlling B. licheniformis spores isolated from dairy materials in yogurt production.

  9. Rapid Detection of Bacillus anthracis Spores Using Immunomagnetic Separation and Amperometry

    Science.gov (United States)

    Waller, David F.; Hew, Brian E.; Holdaway, Charlie; Jen, Michael; Peckham, Gabriel D.

    2016-01-01

    Portable detection and quantitation methods for Bacillus anthracis (anthrax) spores in pure culture or in environmental samples are lacking. Here, an amperometric immunoassay has been developed utilizing immunomagnetic separation to capture the spores and remove potential interferents from test samples followed by amperometric measurement on a field-portable instrument. Antibody-conjugated magnetic beads and antibody-conjugated glucose oxidase were used in a sandwich format for the capture and detection of target spores. Glucose oxidase activity of spore pellets was measured indirectly via amperometry by applying a bias voltage after incubation with glucose, horseradish peroxidase, and the electron mediator 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). Target capture was mediated by polyclonal antisera, whereas monoclonal antibodies were used for signal generation. This strategy maximized sensitivity (500 target spores, 5000 cfu/mL), while also providing a good specificity for Bacillus anthracis spores. Minimal signal deviation occurs in the presence of environmental interferents including soil and modified pH conditions, demonstrating the strengths of immunomagnetic separation. The simultaneous incubation of capture and detection antibodies and rapid substrate development (5 min) result in short sample-to-signal times (less than an hour). With attributes comparable or exceeding that of ELISA and LFDs, amperometry is a low-cost, low-weight, and practical method for detecting anthrax spores in the field. PMID:27999382

  10. Inactivation of Bacillus spores inoculated in milk by Ultra High Pressure Homogenization.

    Science.gov (United States)

    Amador Espejo, Genaro Gustavo; Hernández-Herrero, M M; Juan, B; Trujillo, A J

    2014-12-01

    Ultra High-Pressure Homogenization treatments at 300 MPa with inlet temperatures (Ti) of 55, 65, 75 and 85 °C were applied to commercial Ultra High Temperature treated whole milk inoculated with Bacillus cereus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus coagulans, Geobacillus stearothermophilus and Bacillus subtilis spores in order to evaluate the inactivation level achieved. Ultra High-Pressure Homogenization conditions at 300 MPa with Ti = 75 and 85 °C were capable of a spore inactivation of ∼5 log CFU/mL. Furthermore, under these processing conditions, commercial sterility (evaluated as the complete inactivation of the inoculated spores) was obtained in milk, with the exception of G. stearothermophilus and B. subtilis treated at 300 MPa with Ti = 75 °C. The results showed that G. stearothermophilus and B. subtilis have higher resistance to the Ultra High-Pressure Homogenization treatments applied than the other microorganisms inoculated and that a treatment performed at 300 MPa with Ti = 85 °C was necessary to completely inactivate these microorganisms at the spore level inoculated (∼1 × 10(6) CFU/mL). Besides, a change in the resistance of B. licheniformis, B. sporothermodurans, G. stearothermophilus and B. subtilis spores was observed as the inactivation obtained increased remarkably in treatments performed with Ti between 65 and 75 °C. This study provides important evidence of the suitability of UHPH technology for the inactivation of spores in high numbers, leading to the possibility of obtaining commercially sterile milk.

  11. Novel strategies for enhanced removal of persistent Bacillus anthracis surrogates and Clostridium difficile spores from skin.

    Directory of Open Access Journals (Sweden)

    Michelle M Nerandzic

    Full Text Available 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; P3.5 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.

  12. Structural Analysis of Bacillus subtilis Spore Peptidoglycan during Sporulation

    OpenAIRE

    2000-01-01

    A major structural element of bacterial endospores is a peptidoglycan (PG) wall. This wall is produced between the two opposed membranes surrounding the developing forespore and is composed of two layers. The inner layer is the germ cell wall, which appears to have a structure similar to that of the vegetative cell wall and which serves as the initial cell wall following spore germination. The outer layer, the cortex, has a modified structure, is required for maintenance of spore dehydration,...

  13. Analysis of a Novel Spore Antigen in Bacillus anthracis That Contributes to Spore Opsonization

    Science.gov (United States)

    2008-01-01

    identity with homologues in B. cereus and Bacillus thuringiensis (99 and 94 %, respectively). In addition, a small ORF (BA5270) was located immediately...N. R. (1962). Field evaluation of a human anthrax vaccine. Am J Public Health 52, 632–645. Brossier, F. & Mock, M. (2001). Toxins of Bacillus ...authors (2007). The complete genome sequence of Bacillus thuringiensis Al Hakam. J Bacteriol 189, 3680–3681. Clements, M. O. & Moir, A. (1998). Role of

  14. Comparative analysis of Bacillus weihenstephanensis KBAB4 spores obtained at different temperatures

    NARCIS (Netherlands)

    Garcia, D.; Voort, van der M.; Abee, T.

    2010-01-01

    The impact of Bacillus weihenstephanensis KBAB4 sporulation temperature history was assessed on spore heat resistance, germination and outgrowth capacity at a temperature range from 7 to 30 °C. Sporulation rate and efficiency decreased at low temperature, as cells sporulated at 12, 20 and 30 °C with

  15. Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction

    NARCIS (Netherlands)

    W. Abhyankar; A. ter Beek; H. Dekker; R. Kort; S. Brul; C.G. de Koster

    2011-01-01

    Species from the genus Bacillus have the ability to form endospores, dormant cellular forms that are able to survive heat and acid preservation techniques commonly used in the food industry. Resistance characteristics of spores towards various environmental stresses are in part attributed to their c

  16. Soya bean tempe extracts show antibacterial activity against Bacillus cereus cells and spores

    NARCIS (Netherlands)

    Roubos-van den Hil, P.J.; Dalmas, E.; Nout, M.J.R.; Abee, T.

    2010-01-01

    Aims: Tempe, a Rhizopus ssp.-fermented soya bean food product, was investigated for bacteriostatic and/or bactericidal effects against cells and spores of the food-borne pathogen Bacillus cereus. Methods and results: Tempe extract showed a high antibacterial activity against B. cereus ATCC 14579 bas

  17. Characterization of a spore-specific protein of the Bacillus cereus group

    NARCIS (Netherlands)

    From, C.; Voort, van der M.; Abee, T.; Granum, P.E.

    2012-01-01

    Bc1245 is a monocistronic chromosomal gene of Bacillus cereus ATCC 14579 encoding a putative protein of 143 amino acids identified in this study to have a spore-related function in B. cereus. Bc1245 is highly conserved in the genome of members of the B. cereus group, indicating an important function

  18. Germination and outgrowth of spores of Bacillus cereus group members: diversity and role of germinant receptors.

    Science.gov (United States)

    Abee, Tjakko; Groot, Masja Nierop; Tempelaars, Marcel; Zwietering, Marcel; Moezelaar, Roy; van der Voort, Menno

    2011-04-01

    Bacillus cereus is a gram-positive, facultative anaerobic, endospore-forming toxicogenic human pathogen. Endospores are highly specialized, metabolically dormant cell types that are resistant to extreme environmental conditions, including heat, dehydration and other physical stresses. B. cereus can enter a range of environments, and can in its spore form, survive harsh conditions. If these conditions become favorable, spores can germinate and grow out and reach considerable numbers in a range of environments including processed foods. Certainly the last decade, when consumer preferences have shifted to mildly processed food, new opportunities arose for spore-forming spoilage and pathogenic organisms. Only rigorous methods have been shown to be capable of destroying all spores present in food, thus a shift toward e.g., milder heat preservation strategies, may result in low but significant amounts of viable spores in food products. Hence, the need for a mild spore destruction strategy is eminent including control of spore outgrowth. Consequently, there is a large interest in triggering spore germination in foodstuffs, since germinated spores have lost the extreme resistance of dormant spores and are relatively easy to kill. Another option could be to prevent germination so that no dangerous levels can be reached. This contribution will focus on germination and outgrowth characteristics of B. cereus and other members of the B. cereus group, providing an overview of the niches these spore-formers can occupy, the signals that trigger germination, and how B. cereus copes with these wake-up calls in different environments including foods, during food processing and upon interaction with the human host.

  19. Morphological and mechanical imaging of Bacillus cereus spore formation at the nanoscale.

    Science.gov (United States)

    Wang, Congzhou; Stanciu, Cristina; Ehrhardt, Christopher J; Yadavalli, Vamsi K

    2015-04-01

    Bacteria from the genus Bacillus are able to transform into metabolically dormant states called (endo) spores in response to nutrient deprivation and other harsh conditions. These morphologically distinct spores are fascinating constructs, amongst the most durable cells in nature, and have attracted attention owing to their relevance in food-related illnesses and bioterrorism. Observing the course of bacterial spore formation (sporulation) spatially, temporally and mechanically, from the vegetative cell to a mature spore, is critical for a better understanding of this process. Here, we present a fast and versatile strategy for monitoring both the morphological and mechanical changes of Bacillus cereus bacteria at the nanoscale using atomic force microscopy. Through a strategy of imaging and nanomechanical mapping, we show the morphogenesis of the endospore and released mature endospore. Finally, we investigate individual spores to characterize their surface mechanically. The progression in elasticity coupled with a similarity of characteristic distributions between the incipient endospores and the formed spores show these distinct stages. Taken together, our data demonstrates the power of atomic force microscopy applied in microbiology for probing this important biological process at the single cell scale.

  20. Thermal inactivation of Bacillus anthracis surrogate spores in a bench-scale enclosed landfill gas flare.

    Science.gov (United States)

    Tufts, Jenia A McBrian; Rosati, Jacky A

    2012-02-01

    A bench-scale landfill flare system was designed and built to test the potential for landfilled biological spores that migrate from the waste into the landfill gas to pass through the flare and exit into the environment as viable. The residence times and temperatures of the flare were characterized and compared to full-scale systems. Geobacillus stearothermophilus and Bacillus atrophaeus, nonpathogenic spores that may serve as surrogates for Bacillus anthracis, the causative agent for anthrax, were investigated to determine whether these organisms would be inactivated or remain viable after passing through a simulated landfill flare. High concentration spore solutions were aerosolized, dried, and sent through a bench-scale system to simulate the fate of biological weapon (BW)-grade spores in a landfill gas flare. Sampling was conducted downstream of the flare using a bioaerosol collection device containing sterile white mineral oil. The samples were cultured, incubated for seven days, and assessed for viability. Results showed that the bench-scale system exhibited good similarity to the real-world conditions of an enclosed standard combustor flare stack with a single orifice, forced-draft diffusion burner. All spores of G. stearothermophilus and B. atrophaeus were inactivated in the flare, indicating that spores that become re-entrained in landfill gas may not escape the landfill as viable, apparently becoming completely inactivated as they exit through a landfill flare.

  1. Evaluation of germination, distribution, and persistence of Bacillus subtilis spores through the gastrointestinal tract of chickens.

    Science.gov (United States)

    Latorre, J D; Hernandez-Velasco, X; Kallapura, G; Menconi, A; Pumford, N R; Morgan, M J; Layton, S L; Bielke, L R; Hargis, B M; Téllez, G

    2014-07-01

    Spores are popular as direct-fed microbials, though little is known about their mode of action. Hence, the first objective of the present study was to evaluate the in vitro germination and growth rate of Bacillus subtilis spores. Approximately 90% of B. subtilis spores germinate within 60 min in the presence of feed in vitro. The second objective was to determine the distribution of these spores throughout different anatomical segments of the gastrointestinal tract (GIT) in a chicken model. For in vivo evaluation of persistence and dissemination, spores were administered to day-of-hatch broiler chicks either as a single gavage dose or constantly in the feed. During 2 independent experiments, chicks were housed in isolation chambers and fed sterile corn-soy-based diets. In these experiments one group of chickens was supplemented with 10(6) spores/g of feed, whereas a second group was gavaged with a single dose of 10(6) spores per chick on day of hatch. In both experiments, crop, ileum, and cecae were sampled from 5 chicks at 24, 48, 72, 96, and 120 h. Viable B. subtilis spores were determined by plate count method after heat treatment (75°C for 10 min). The number of recovered spores was constant through 120 h in each of the enteric regions from chickens receiving spores supplemented in the feed. However, the number of recovered B. subtilis spores was consistently about 10(5) spores per gram of digesta, which is about a 1-log10 reduction of the feed inclusion rate, suggesting approximately a 90% germination rate in the GIT when fed. On the other hand, recovered B. subtilis spores from chicks that received a single gavage dose decreased with time, with only approximately 10(2) spores per gram of sample by 120 h. This confirms that B. subtilis spores are transiently present in the GIT of chickens, but the persistence of vegetative cells is presently unknown. For persistent benefit, continuous administration of effective B. subtilis direct-fed microbials as vegetative

  2. Bacillus globigii bugbeads: a model simulant of a bacterial spore.

    Science.gov (United States)

    Farrell, Svetlana; Halsall, H Brian; Heineman, William R

    2005-01-15

    Nonpathogenic microorganisms are often used as simulants of biological pathogens during the initial phase of detection method development. While these simulants approximate the size, shape, and cellular organization of the microorganism of interest, they do not resemble its surface protein content, a factor particularly important in methods based on immunorecognition. Here, we develop and detect an artificial bacterial spore--B. globigii (BG) Bugbead-a particle mimicking the antigenic surface of BG spores. Two methods of spore protein extraction were compared both quantitatively (by protein concentration assay) and qualitatively (by SDS-PAGE and Western blot): extraction by mechanical disruption and extraction by chemical decoating. The former method was more efficient in producing more protein and a greater number of antigens. BG Bugbeads were made by conjugating the extracted proteins to 0.8-microm carboxyl-coated polystyrene particles via carbodiimide coupling. BG Bugbeads were successfully detected by a bead-based enzyme-labeled immunoassay with fluorescence detection with a detection limit of 6.9 x 10(3) particles/mL. Formation of the Bugbead-capture bead complex was confirmed by ESEM. The concept of a harmless artificial spore can be applied to developing improved simulants for pathogenic spore-forming microorganisms such as B. anthracis, C. botulinum, and B. cereus, which can to be used for method validation, instrument calibration, and troubleshooting.

  3. Bacillus cereus spores and cereulide in food-borne illness

    OpenAIRE

    Shaheen, Ranad

    2009-01-01

    B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≀ pH 1) e...

  4. Graphical procedure for comparing thermal death of Bacillus stearothermophilus spores in saturated and superheated steam.

    Science.gov (United States)

    SHULL, J J; ERNST, R R

    1962-09-01

    The thermal death curve of dried spores of Bacillus stearothermophilus in saturated steam was characterized by three phases: (i) a sharp initial rise in viable count; (ii) a low rate of death which gradually increased; and (iii) logarithmic death at maximal rate. The first phase was a reflection of inadequate heat activation of the spore population. The second and third phases represented the characteristic thermal death curve of the spores in saturated steam. A jacketed steam sterilizer, equipped with a system for initial evacuation of the chamber, was examined for superheat during normal operation. Measurements of spore inactivation and temperature revealed superheat in surface layers of fabrics being processed in steam at 121 C. The high temperature of the fabric surfaces was attributed to absorption of excess heat energy from superheated steam. The superheated steam was produced at the beginning of the normal sterilizing cycle by transfer of heat from the steam-heated jacket to saturated steam entering the vessel.

  5. Investigating the thermodynamic stability of Bacillus subtilis spore-uranium(VI) adsorption though surface complexation modeling

    Science.gov (United States)

    Harrold, Z.; Hertel, M.; Gorman-Lewis, D.

    2012-12-01

    Dissolved uranium speciation, mobility, and remediation are increasingly important topics given continued and potential uranium (U) release from mining operations and nuclear waste. Vegetative bacterial cell surfaces are known to adsorb uranium and may influence uranium speciation in the environment. Previous investigations regarding U(VI) adsorption to bacterial spores, a differentiated and dormant cell type with a tough proteinaceous coat, include U adsorption affinity and XAFS data. We investigated the thermodynamic stability of aerobic, pH dependent uranium adsorption to bacterial spore surfaces using purified Bacillus subtilis spores in solution with 5ppm uranium. Adsorption reversibility and kinetic experiments indicate that uranium does not precipitate over the duration of the experiments and equilibrium is reached within 20 minutes. Uranium-spore adsorption edges exhibited adsorption at all pH measured between 2 and 10. Maximum adsorption was achieved around pH 7 and decreased as pH increased above 7. We used surface complexation modeling (SCM) to quantify uranium adsorption based on balanced chemical equations and derive thermodynamic stability constants for discrete uranium-spore adsorption reactions. Site specific thermodynamic stability constants provide insight on interactions occurring between aqueous uranium species and spore surface ligands. The uranium adsorption data and SCM parameters described herein, also provide a basis for predicting the influence of bacterial spores on uranium speciation in natural systems and investigating their potential as biosorption agents in engineered systems.

  6. Improved proteomic analysis following trichloroacetic acid extraction of Bacillus anthracis spore proteins.

    Science.gov (United States)

    Deatherage Kaiser, Brooke L; Wunschel, David S; Sydor, Michael A; Warner, Marvin G; Wahl, Karen L; Hutchison, Janine R

    2015-11-01

    Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Analysis of cellular proteins is dependent upon efficient extraction from bacterial samples, which can be challenging with increasing complexity and refractory characteristics. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrichment for certain classes of proteins. The method presented here is technically simple, does not require specialized equipment such as a mechanical disrupter, and is effective for protein extraction of the particularly challenging sample type of Bacillus anthracis Sterne spores. The ability of Trichloroacetic acid (TCA) extraction to isolate proteins from spores and enrich for spore-specific proteins was compared to the traditional mechanical disruption method of bead beating. TCA extraction improved the total average number of proteins identified within a sample as compared to bead beating (547 vs 495, respectively). Further, TCA extraction enriched for 270 spore proteins, including those typically identified by first isolating the spore coat and exosporium layers. Bead beating enriched for 156 spore proteins more typically identified from whole spore proteome analyses. The total average number of proteins identified was equal using TCA or bead beating for easily lysed samples, such as B. anthracis vegetative cells. As with all assays, supplemental methods such as implementation of an alternative preparation method may simplify sample preparation and provide additional insight to the protein biology of the organism being studied.

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

    Science.gov (United States)

    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.

  8. Storage Effects on Sample Integrity of Environmental Surface Sampling Specimens with Bacillus anthracis Spores.

    Science.gov (United States)

    Perry, K Allison; O'Connell, Heather A; Rose, Laura J; Noble-Wang, Judith A; Arduino, Matthew J

    The effect of packaging, shipping temperatures and storage times on recovery of Bacillus anthracis. Sterne spores from swabs was investigated. Macrofoam swabs were pre-moistened, inoculated with Bacillus anthracis spores, and packaged in primary containment or secondary containment before storage at -15°C, 5°C, 21°C, or 35°C for 0-7 days. Swabs were processed according to validated Centers for Disease Control/Laboratory Response Network culture protocols, and the percent recovery relative to a reference sample (T0) was determined for each variable. No differences were observed in recovery between swabs held at -15° and 5°C, (p ≥ 0.23). These two temperatures provided significantly better recovery than swabs held at 21°C or 35°C (all 7 days pooled, p ≤ 0.04). The percent recovery at 5°C was not significantly different if processed on days 1, 2 or 4, but was significantly lower on day 7 (day 2 vs. 7, 5°C, 10(2), p=0.03). Secondary containment provided significantly better percent recovery than primary containment, regardless of storage time (5°C data, p ≤ 0.008). The integrity of environmental swab samples containing Bacillus anthracis spores shipped in secondary containment was maintained when stored at -15°C or 5°C and processed within 4 days to yield the optimum percent recovery of spores.

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

    Energy Technology Data Exchange (ETDEWEB)

    Plomp, M; Leighton, T J; Wheeler, K E; Malkin, A J

    2004-05-06

    The capability to image single microbial cell surfaces at nanometer scale under native conditions would profoundly impact mechanistic and structural studies of pathogenesis, immunobiology, environmental resistance and biotransformation. We report here that advances in atomic force microscopy (AFM) have allowed us to directly visualize high-resolution native structures of bacterial endospores, including the exosporium and spore coats of four Bacillus species in air and water environments. The dimensions of individual Bacillus atrophaeus spores were found to decrease reversibly by 12% in response to a change in the environment from aqueous to aerial phase. Intraspecies spore size distribution analyses revealed that spore length could vary by a factor of 2 while the absolute deviation is 7 - 13% in length and 4 - 6 % in width. AFM analysis also demonstrated that the mechanisms of spore coat self-assembly are similar to those described for inorganic and macromolecular crystallization. These results establish AFM as a powerful new tool for the analysis of molecular architecture and variability as a function of spatial, temporal and developmental organizational scales.

  10. Identification and validation of specific markers of Bacillus anthracis spores by proteomics and genomics approaches.

    Science.gov (United States)

    Chenau, Jérôme; Fenaille, François; Caro, Valérie; Haustant, Michel; Diancourt, Laure; Klee, Silke R; Junot, Christophe; Ezan, Eric; Goossens, Pierre L; Becher, François

    2014-03-01

    Bacillus anthracis is the causative bacteria of anthrax, an acute and often fatal disease in humans. The infectious agent, the spore, represents a real bioterrorism threat and its specific identification is crucial. However, because of the high genomic relatedness within the Bacillus cereus group, it is still a real challenge to identify B. anthracis spores confidently. Mass spectrometry-based tools represent a powerful approach to the efficient discovery and identification of such protein markers. Here we undertook comparative proteomics analyses of Bacillus anthracis, cereus and thuringiensis spores to identify proteoforms unique to B. anthracis. The marker discovery pipeline developed combined peptide- and protein-centric approaches using liquid chromatography coupled to tandem mass spectrometry experiments using a high resolution/high mass accuracy LTQ-Orbitrap instrument. By combining these data with those from complementary bioinformatics approaches, we were able to highlight a dozen novel proteins consistently observed across all the investigated B. anthracis spores while being absent in B. cereus/thuringiensis spores. To further demonstrate the relevance of these markers and their strict specificity to B. anthracis, the number of strains studied was extended to 55, by including closely related strains such as B. thuringiensis 9727, and above all the B. cereus biovar anthracis CI, CA strains that possess pXO1- and pXO2-like plasmids. Under these conditions, the combination of proteomics and genomics approaches confirms the pertinence of 11 markers. Genes encoding these 11 markers are located on the chromosome, which provides additional targets complementary to the commonly used plasmid-encoded markers. Last but not least, we also report the development of a targeted liquid chromatography coupled to tandem mass spectrometry method involving the selection reaction monitoring mode for the monitoring of the 4 most suitable protein markers. Within a proof

  11. Processing, Assembly and Localization of a Bacillus anthracis Spore Protein

    Science.gov (United States)

    2010-01-01

    10.1099/ mic .0.033407-0 174 033407 Printed in Great Britain Approved for public release. Distribution is unlimited Report Documentation Page Form...on LB agar plates to assay viable cells. In vivo challenges. Female Hartley guinea pigs (350–400 g) were obtained from Charles River Laboratories...Guinea pigs were challenged intramuscularly (Fellows et al., 2001) by injection of 200 ml of heat-activated spores suspended in water. The animals were

  12. The use of germinants to potentiate the sensitivity of Bacillus anthracis spores to peracetic acid

    Directory of Open Access Journals (Sweden)

    Ozgur eCelebi

    2016-01-01

    Full Text Available Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM and inosine (5 mM to reduce the concentration of peracetic acid (PAA required to inactivate B.anthracis spores. While L-alanine significantly enhanced (p=0.0085 the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p=0.0009. To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B.anthracis to increase the level of contamination to 104 spores/g. Treatment with germinants followed one hour later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B.anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p<0.0001 in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B.anthracis spores contaminated sites.

  13. Involvement of Coat Proteins in Bacillus subtilis Spore Germination in High-Salinity Environments.

    Science.gov (United States)

    Nagler, Katja; Setlow, Peter; Reineke, Kai; Driks, Adam; Moeller, Ralf

    2015-10-01

    The germination of spore-forming bacteria in high-salinity environments is of applied interest for food microbiology and soil ecology. It has previously been shown that high salt concentrations detrimentally affect Bacillus subtilis spore germination, rendering this process slower and less efficient. The mechanistic details of these salt effects, however, remained obscure. Since initiation of nutrient germination first requires germinant passage through the spores' protective integuments, the aim of this study was to elucidate the role of the proteinaceous spore coat in germination in high-salinity environments. Spores lacking major layers of the coat due to chemical decoating or mutation germinated much worse in the presence of NaCl than untreated wild-type spores at comparable salinities. However, the absence of the crust, the absence of some individual nonmorphogenetic proteins, and the absence of either CwlJ or SleB had no or little effect on germination in high-salinity environments. Although the germination of spores lacking GerP (which is assumed to facilitate germinant flow through the coat) was generally less efficient than the germination of wild-type spores, the presence of up to 2.4 M NaCl enhanced the germination of these mutant spores. Interestingly, nutrient-independent germination by high pressure was also inhibited by NaCl. Taken together, these results suggest that (i) the coat has a protective function during germination in high-salinity environments; (ii) germination inhibition by NaCl is probably not exerted at the level of cortex hydrolysis, germinant accessibility, or germinant-receptor binding; and (iii) the most likely germination processes to be inhibited by NaCl are ion, Ca(2+)-dipicolinic acid, and water fluxes.

  14. The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid.

    Science.gov (United States)

    Celebi, Ozgur; Buyuk, Fatih; Pottage, Tom; Crook, Ant; Hawkey, Suzanna; Cooper, Callum; Bennett, Allan; Sahin, Mitat; Baillie, Leslie

    2016-01-01

    Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 10(4) spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites.

  15. HtrC is involved in proteolysis of YpeB during germination of Bacillus anthracis and Bacillus subtilis spores.

    Science.gov (United States)

    Bernhards, Casey B; Chen, Yan; Toutkoushian, Hannah; Popham, David L

    2015-01-01

    Bacterial endospores can remain dormant for decades yet can respond to nutrients, germinate, and resume growth within minutes. An essential step in the germination process is degradation of the spore cortex peptidoglycan wall, and the SleB protein in Bacillus species plays a key role in this process. Stable incorporation of SleB into the spore requires the YpeB protein, and some evidence suggests that the two proteins interact within the dormant spore. Early during germination, YpeB is proteolytically processed to a stable fragment. In this work, the primary sites of YpeB cleavage were identified in Bacillus anthracis, and it was shown that the stable products are comprised of the C-terminal domain of YpeB. Modification of the predominant YpeB cleavage sites reduced proteolysis, but cleavage at other sites still resulted in loss of full-length YpeB. A B. anthracis strain lacking the HtrC protease did not generate the same stable YpeB products. In B. anthracis and Bacillus subtilis htrC mutants, YpeB was partially stabilized during germination but was still degraded at a reduced rate by other, unidentified proteases. Purified HtrC cleaved YpeB to a fragment similar to that observed in vivo, and this cleavage was stimulated by Mn(2+) or Ca(2+) ions. A lack of HtrC did not stabilize YpeB or SleB during spore formation in the absence of the partner protein, indicating other proteases are involved in their degradation during sporulation.

  16. Persistence strategies of Bacillus cereus spores isolated from dairy silo tanks.

    Science.gov (United States)

    Shaheen, Ranad; Svensson, Birgitta; Andersson, Maria A; Christiansson, Anders; Salkinoja-Salonen, Mirja

    2010-05-01

    Survival of Bacillus cereus spores of dairy silo tank origin was investigated under conditions simulating those in operational dairy silos. Twenty-three strains were selected to represent all B. cereus isolates (n = 457) with genotypes (RAPD-PCR) that frequently colonised the silo tanks of at least two of the sampled eight dairies. The spores were studied for survival when immersed in liquids used for cleaning-in-place (1.0% sodium hydroxide at pH 13.1, 75 degrees C; 0.9% nitric acid at pH 0.8, 65 degrees C), for adhesion onto nonliving surfaces at 4 degrees C and for germination and biofilm formation in milk. Four groups with different strategies for survival were identified. First, high survival (log 15 min kill steel from cold water. Third, a cereulide producing group with spores characterised by slow germination in rich medium and well preserved viability when exposed to heating at 90 degrees C. Fourth, spores capable of germinating at 8 degrees C and possessing the cspA gene. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot-alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus.

  17. Spatially resolved characterization of water and ion incorporation in Bacillus spores.

    Science.gov (United States)

    Ghosal, Sutapa; Leighton, Terrance J; Wheeler, Katherine E; Hutcheon, Ian D; Weber, Peter K

    2010-05-01

    We present the first direct visualization and quantification of water and ion uptake into the core of individual dormant Bacillus thuringiensis subsp. israelensis (B. thuringiensis subsp. israelensis) endospores. Isotopic and elemental gradients in the B. thuringiensis subsp. israelensis spores show the permeation and incorporation of deuterium in deuterated water (D(2)O) and solvated ions throughout individual spores, including the spore core. Under hydrated conditions, incorporation into a spore occurs on a time scale of minutes, with subsequent uptake of the permeating species continuing over a period of days. The distribution of available adsorption sites is shown to vary with the permeating species. Adsorption sites for Li(+), Cs(+), and Cl(-) are more abundant within the spore outer structures (exosporium, coat, and cortex) relative to the core, while F(-) adsorption sites are more abundant in the core. The results presented here demonstrate that elemental abundance and distribution in dormant spores are influenced by the ambient environment. As such, this study highlights the importance of understanding how microbial elemental and isotopic signatures can be altered postproduction, including during sample preparation for analysis, and therefore, this study is immediately relevant to the use of elemental and isotopic markers in environmental microbiology and microbial forensics.

  18. Evaluation of a Stochastic Inactivation Model for Heat-Activated Spores of Bacillus spp. ▿

    Science.gov (United States)

    Corradini, Maria G.; Normand, Mark D.; Eisenberg, Murray; Peleg, Micha

    2010-01-01

    Heat activates the dormant spores of certain Bacillus spp., which is reflected in the “activation shoulder” in their survival curves. At the same time, heat also inactivates the already active and just activated spores, as well as those still dormant. A stochastic model based on progressively changing probabilities of activation and inactivation can describe this phenomenon. The model is presented in a fully probabilistic discrete form for individual and small groups of spores and as a semicontinuous deterministic model for large spore populations. The same underlying algorithm applies to both isothermal and dynamic heat treatments. Its construction does not require the assumption of the activation and inactivation kinetics or knowledge of their biophysical and biochemical mechanisms. A simplified version of the semicontinuous model was used to simulate survival curves with the activation shoulder that are reminiscent of experimental curves reported in the literature. The model is not intended to replace current models to predict dynamic inactivation but only to offer a conceptual alternative to their interpretation. Nevertheless, by linking the survival curve's shape to probabilities of events at the individual spore level, the model explains, and can be used to simulate, the irregular activation and survival patterns of individual and small groups of spores, which might be involved in food poisoning and spoilage. PMID:20453137

  19. Dry heat exposures of surface exposed and embedded Bacillus spores

    Science.gov (United States)

    Schubert, Wayne

    Dry heat microbial reduction (DHMR) is the primary technique used to reduce the microbial load of spacecraft and component parts. Often, manufacturing procedures require heating flight hardware to high temperatures for purposes other than planetary protection DHMR. The existing specifications, however, do not allow for additional planetary protection bioburden reduction credit if the hardware is exposed without controlled relative humidity. The intent of this study was to provide adequate data on the DHMR technique to support modification of four aspects of current requirements; expansion of acceptable time and temperature combinations used for spacecraft dry heat microbial reduction processes above 125° C, determining the effect that humidity has on spore lethality as a function of temperature, understanding the lethality for spores with exceptionally high thermal resistance and to investigate the extended exposure requirement for materials that might contain embedded microorganisms. Spores from two bacterial species were tested, B. atrophaeus ATCC 9372 and B. sp. ATCC 29669, under three conditions encompassing 5 temperature points. Embedded experiments utilized a silicone rubber polymer that is commonly used on robotic spacecraft, and surface exposed experiments were performed under both ambient and vacuum-controlled humidity conditions. The results obtained support the use of DHMR protocols that extend the maximum temperature range from 125° C to 170° C, with either controlled or ambient humidity. If implemented, this will give projects bioburden reduction credit for shorter treatments at extended temperatures, and allow spacecraft to be processed in more readily available and less expensive facilities that do not have humidity control, with significant cost and schedule benefits. The study also demonstrated that the required heating time for materials presumed to have embedded bioburden is conservative.

  20. Development of a Rapid and Sensitive Immunoassay for Detection and Subsequent Recovery of Bacillus anthracis Spores in Environmental Samples

    Science.gov (United States)

    Bacillus anthracis is considered a major threat as an agent of bioterrorism. B. anthracis spores are readily dispersed as aerosols, are very persistent, and are resistant to normal disinfection treatments. Immunoassays have been developed to rapidly detect B. anthracis spores at high concentration...

  1. Sporulation environment of emetic toxin-producing Bacillus cereus strains determines spore size, heat resistance and germination capacity

    NARCIS (Netherlands)

    Voort, van der M.; Abee, T.

    2013-01-01

    Aim Heat resistance, germination and outgrowth capacity of Bacillus cereus spores in processed foods are major factors in causing the emetic type of gastrointestinal disease. In this study, we aim to identify the impact of different sporulation conditions on spore properties of emetic toxin-producin

  2. The Adsorption Properties of Bacillus atrophaeus Spores on Single-Wall Carbon Nanotubes

    OpenAIRE

    Cortes, P; S. Deng; Smith, G. B.

    2009-01-01

    An adsorption equilibrium and a kinetic study of Bacillus atrophaeus on Single-Wall Carbon Nanotubes (SWCNTs) were here performed to provide the basis for developing biosensor devices for detecting threatening micro-organisms in water supply systems. B. atrophaeus spores and carbon nanotubes were subjected to a batch adsorption process to document their equilibria and kinetics. Here, commercial nanotubes were either studied as received or were acid-purified before adsorption experiments. The ...

  3. Spore and crystal formation in Bacillus thuringiensis var thuringiensis during growth in cystine and cysteine.

    OpenAIRE

    Rajalakshmi, S.; Shethna, YI

    1980-01-01

    The effect of the addition of different concentratons of cystine and cysteine on sporulation and parasporal crystal formation in Bacillus thuringiensis var. thuringiensis was studied. The effect was well pronounced when the systine/cysteine additions were made after the stationary phase. Heat stable spores and crystals were formed when the culture was provided with a low concentration of cystine/cysteine (0.05 per cent w/v). At a moderate concentration of cystine or cysteine (0.15%), only ...

  4. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways

    Energy Technology Data Exchange (ETDEWEB)

    Kabilan, S.; Suffield, S. R.; Recknagle, K. P.; Jacob, R. E.; Einstein, D. R.; Kuprat, A. P.; Carson, J. P.; Colby, S. M.; Saunders, J. H.; Hines, S. A.; Teeguarden, J. G.; Straub, T. M.; Moe, M.; Taft, S. C.; Corley, R. A.

    2016-09-01

    Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived respectively from computed tomography (CT) and µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation–exhalation breathing conditions using average species-specific minute volumes. Two different exposure scenarios were modeled in the rabbit based upon experimental inhalation studies. For comparison, human simulations were conducted at the highest exposure concentration used during the rabbit experimental exposures. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the nasal sinus compared to the human at the same air concentration of anthrax spores. In contrast, higher spore deposition was predicted in the lower conducting airways of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology for deposition.

  5. Detection of Bacillus spores within 15 minutes by surface-enhanced Raman spectroscopy

    Science.gov (United States)

    Shende, Chetan; Inscore, Frank; Huang, Hermes; Farquharson, Stuart; Sengupta, Atanu

    2012-06-01

    Since the distribution of Bacillus anthracis causing spores through the US Postal System, there has been a persistent fear that biological warfare agents (BWAs) will be used by terrorists against our military abroad and our civilians at home. Despite the substantial effort to develop BWA analyzers, they remain either too slow, produce high falsealarm rates, lack sensitivity, or cannot be fielded. Consequently there remains a need for a portable analyzer that can overcome these limitations as expressed at the 2011 Biological Weapons Convention. To meet this need we have been developing a sample system that selectively binds BWAs and produce surface-enhanced Raman (SER) spectra using portable Raman spectrometers. Here we describe the use of a short peptide ligand functionalized on silver nanoparticles to selectively capture Bacillus cereus spores (a surrogate of B. anthracis) and their subsequent detection by SER spectroscopy. This technique was used to specifically detect B. cereus spores over closely related species like B. subtilis belonging to the same genus within 15 minutes. Sensitivity of the method was demonstrated by detecting 104 B. cereus spores/mL of water. The technology, once developed should prove invaluable for rapid monitoring of BWAs, which will immensely help first responders and emergency personnel in implementing appropriate counter measures.

  6. Bacillus spores as building blocks for stimuli-responsive materials and nanogenerators

    Science.gov (United States)

    Sahin, Ozgur; Chen, Xi

    2014-03-01

    Materials that mechanically respond to external chemical stimuli have applications in a wide range of fields. Inspired by biological systems, stimuli-responsive materials that can oscillate, transport fluid, mimic homeostasis, and undergo complex changes in shape have been previously demonstrated. However, the effectiveness of synthetic stimuli-responsive materials in generating work is limited when compared to mechanical actuators. During studies of bacterial sporulation, we have found that the mechanical response of Bacillus spores to water gradients exhibits an energy density of more than 10 MJ/m3, which is two orders of magnitude higher than synthetic water-responsive materials. We also identified mutations that can approximately double the energy density of the spores, and found that spores can self-assemble into dense, submicron-thick monolayers on substrates such as silicon microcantilevers and elastomer sheets, creating self-assembled actuators that can remotely generate electrical power from an evaporating body of water. The energy conversion mechanism of Bacillus spores may facilitate synthetic stimuli-responsive materials with significantly higher energy densities. We acknowledge support from the U.S. Dept. of Energy Early Career Research Program, the Wyss Institute for Biologically Inspired Engineering, and the Rowland Institute at Harvard.

  7. Disinfection methods for spores of Bacillus atrophaeus, B. anthracis, Clostridium tetani, C. botulinum and C. difficile.

    Science.gov (United States)

    Oie, Shigeharu; Obayashi, Akiko; Yamasaki, Hirofumi; Furukawa, Hiroyuki; Kenri, Tsuyoshi; Takahashi, Motohide; Kawamoto, Keiko; Makino, Sou-ichi

    2011-01-01

    To evaluate disinfection methods for environments contaminated with bioterrorism-associated microorganism (Bacillus anthracis), we performed the following experiments. First, the sporicidal effects of sodium hypochlorite on spores of five bacterial species were evaluated. Bacillus atrophaeus was the most resistant to hypochlorite, followed in order by B. anthracis, Clostridium botulinum and Clostridium tetani, and Clostridium difficile. Subsequently, using B. atrophaeus spores that were the most resistant to hypochlorite, the sporicidal effects of hypochlorite at lower pH by adding vinegar were evaluated. Hypochlorite containing vinegar had far more marked sporicidal effects than hypochlorite alone. Cleaning with 0.5% (5000 ppm) hypochlorite containing vinegar inactivated B. atrophaeus spores attached to vinyl chloride and plywood plates within 15 s, while that not containing vinegar did not inactivate spores attached to cement or plywood plates even after 1 h. Therefore, the surfaces of cement or plywood plates were covered with gauze soaked in 0.5% hypochlorite containing vinegar, and the sporicidal effects were evaluated. B. atrophaeus spores attached to plywood plates were not inactivated even after 6 h, but those attached to cement plates were inactivated within 5 min. On the other hand, covering the surfaces of plywood plates with gauze soaked in 0.3% peracetic acid and gauze soaked in 2% glutaral inactivated B. atrophaeus spores within 5 min and 6 h, respectively. These results suggest that hypochlorite containing vinegar is effective for disinfecting vinyl chloride, tile, and cement plates contaminated with B. anthracis, and peracetic acid is effective for disinfecting plywood plates contaminated with such microorganism.

  8. Carbon-13 (13C) labeling of Bacillus subtilis vegetative cells and spores: suitability for DNA stable isotope probing (DNA-SIP) of spores in soils.

    Science.gov (United States)

    Nicholson, Wayne L; Fedenko, Jeffrey; Schuerger, Andrew C

    2009-07-01

    To test the suitability of DNA stable isotope probing (DNA-SIP) for characterizing bacterial spore populations in soils, the properties of Bacillus subtilis cells and spores intensely labeled with [(13)C]glucose were characterized. Spore germination, vegetative growth rates, and sporulation efficiency were indistinguishable on glucose versus [(13)C]glucose, as were spore wet heat and UV resistance. Unlabeled and (13)C-labeled spores contained 1.0989 and 74.336 at.% (13)C, and exhibited wet densities of 1.356 and 1.365 g/ml, respectively. Chromosomal DNAs containing (12)C versus (13)C were readily separated by their different buoyant densities in cesium chloride/ethidium bromide gradients.

  9. Mapping of Proteomic Composition on the Surfaces of Bacillus spores by Atomic Force Microscopy-based Immunolabeling

    Energy Technology Data Exchange (ETDEWEB)

    Plomp, M; Malkin, A J

    2008-06-02

    Atomic force microscopy provides a unique capability to image high-resolution architecture and structural dynamics of pathogens (e.g. viruses, bacteria and bacterial spores) at near molecular resolution in native conditions. Further development of atomic force microscopy in order to enable the correlation of pathogen protein surface structures with specific gene products is essential to understand the mechanisms of the pathogen life cycle. We have applied an AFM-based immunolabeling technique for the proteomic mapping of macromolecular structures through the visualization of the binding of antibodies, conjugated with nanogold particles, to specific epitopes on Bacillus spore surfaces. This information is generated while simultaneously acquiring the surface morphology of the pathogen. The immunospecificity of this labeling method was established through the utilization of specific polyclonal and monoclonal antibodies that target spore coat and exosporium epitopes of Bacillus atrophaeus and Bacillus anthracis spores.

  10. Computational Fluid Dynamics Modeling of Bacillus anthracis Spore Deposition in Rabbit and Human Respiratory Airways

    Energy Technology Data Exchange (ETDEWEB)

    Kabilan, Senthil; Suffield, Sarah R.; Recknagle, Kurtis P.; Jacob, Rick E.; Einstein, Daniel R.; Kuprat, Andrew P.; Carson, James P.; Colby, Sean M.; Saunders, James H.; Hines, Stephanie; Teeguarden, Justin G.; Straub, Tim M.; Moe, M.; Taft, Sarah; Corley, Richard A.

    2016-09-30

    Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived from computed tomography (CT) or µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation-exhalation breathing conditions using average species-specific minute volumes. The highest exposure concentration was modeled in the rabbit based upon prior acute inhalation studies. For comparison, human simulation was also conducted at the same concentration. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the upper conducting airways compared to the human at the same air concentration of anthrax spores. As a result, higher particle deposition was predicted in the conducting airways and deep lung of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology.

  11. Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations.

    Science.gov (United States)

    Mansour, M; Amri, D; Bouttefroy, A; Linder, M; Milliere, J B

    1999-02-01

    The effects of nisin and monolaurin, alone and in combination, were investigated on Bacillus licheniformis spores in milk at 37 degrees C. In the absence of inhibitors, germinated spores developed into growing vegetative cells and started sporulation at the end of the exponential phase. In the presence of nisin (25 IU ml-1), spore outgrowth was inhibited (4 log10 reduction at 10 h). Regrowth appeared between 10 and 24 h and reached a high population level (1.25 x 10(8) cfu ml-1) after 7 d. Monolaurin (250 micrograms ml-1) had a bacteriostatic effect during the first 10 h but thereafter, regrowth occurred slowly with a population level after 7 d (4 x 10(5) cfu ml-1) lower than that of nisin. Different combined effects of nisin (between 0 and 42 IU ml-1), monolaurin (ranging from 0 to 300 micrograms ml-1), pH values (between 5.0 and 7.0) and spore loads (10(3), 10(4), 10(5) spores ml-1) were investigated using a Doehlert matrix in order to study the main effects of these factors and the different interactions. Results were analysed using the Response Surface Methodology (RSM) and indicated that nisin and monolaurin had no action on spores before germination; only pH values had a significant effect (P monolaurin (100 micrograms ml-1) in combination acted synergistically on outgrown spores and vegetative cells, showing total inhibition at pH 6.0, without regrowth, within 7 d at 37 degrees C.

  12. Sterilization effect of UV light on Bacillus spores using TiO2 films depends on wavelength

    OpenAIRE

    Nhung, Le Thi Tuyet; Nagata, Hirofumi; Takahashi, Akira; Aihara, Mutsumi; Okamoto, Toshihiro; Shimohata, Takaaki; Mawatari, Kazuaki; Akutagawa, Masatake; Kinouchi, Yohsuke; Haraguchi, Masanobu

    2012-01-01

    UV light and photocatalysts such as titanium dioxide (TiO2) and silver (Ag) are useful for disinfection of water and surfaces. However, the effect of UV wavelength on photocatalytic disinfection of spores is not well understood. Inactivation of Bacillus spores has been examined using different UV wavelengths and TiO2 or TiO2/Ag composite materials. The level of UVA disinfection of Bacillus anthracis and Bacillus brevis vegetative cells increased with the presence of the TiO2 and Ag photocatal...

  13. Rapid detection of Bacillus anthracis spores using a super-paramagnetic lateral-flow immunological detection system.

    Science.gov (United States)

    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.

  14. On the origin of heterogeneity in (preservation) resistance of Bacillus spores: Input for a ‘systems’ analysis approach of bacterial spore outgrowth

    NARCIS (Netherlands)

    Hornstra, L.M.; ter Beek, A.; Smelt, J.P.; Kallemeijn, W.W.; Brul, S.

    2009-01-01

    Bacterial spores are the ultimate (stress) ‘survival capsules’. They allow strains from the Bacillus and Clostridium species to survive harsh environmental conditions. In addition to the decision to enter sporulation the decision to do the reverse (germinate) is also a decisive event after which the

  15. Spore prevalence and toxigenicity of Bacillus cereus and Bacillus thuringiensis isolates from U.S. retail spices.

    Science.gov (United States)

    Hariram, Upasana; Labbé, Ronald

    2015-03-01

    Recent incidents of foodborne illness associated with spices as the vehicle of transmission prompted this examination of U.S. retail spices with regard to Bacillus cereus. This study focused on the levels of aerobic-mesophilic spore-forming bacteria and B cereus spores associated with 247 retail spices purchased from five states in the United States. Samples contained a wide range of aerobic-mesophilic bacterial spore counts ( 10(7) CFU/g). Using a novel chromogenic agar, B. cereus and B. thuringiensis spores were isolated from 77 (31%) and 11 (4%) samples, respectively. Levels of B. cereus were thuringiensis isolates possessed at least one type of enterotoxin gene: HBL (hemolysin BL) or nonhemolytic enterotoxin (NHE). None of the 88 isolates obtained in this study possessed the emetic toxin gene (ces). Using commercially available immunological toxin detection kits, the toxigenicity of the isolates was confirmed. The NHE enterotoxin was expressed in 98% of B. cereus and 91% of B. thuringiensis isolates that possessed the responsible gene. HBL enterotoxin was detected in 87% of B. cereus and 100% of B. thuringiensis PCR-positive isolates. Fifty-two percent of B. cereus and 54% of B. thuringiensis isolates produced both enterotoxins. Ninety-seven percent of B. cereus isolates grew at 12°C, although only two isolates grew well at 9°C. The ability of these spice isolates to form spores, produce diarrheal toxins, and grow at moderately abusive temperatures makes retail spices an important potential vehicle for foodborne illness caused by B. cereus strains, in particular those that produce diarrheal toxins.

  16. An improved system for the surface immobilisation of proteins on Bacillus thuringiensis vegetative cells and spores through a new spore cortex-lytic enzyme anchor.

    Science.gov (United States)

    Shao, Xiaohu; Ni, Hong; Lu, Ting; Jiang, Mengtian; Li, Hua; Huang, Xinfeng; Li, Lin

    2012-02-15

    An improved surface-immobilisation system was engineered to target heterologous proteins onto vegetative cells and spores of Bacillus thuringiensis plasmid-free recipient strain BMB171. The sporulation-dependent spore cortex-lytic enzyme from B. thuringiensis YBT-1520, SceA, was expressed in vegetative cells and used as the surface anchoring motif. Green fluorescent protein (GFP) and a Bacillus endo-β-1,3-1,4-glucanase (BglS) were used as the fusion partners to test the binding efficiency and the functional activities of immobilised surface proteins. The surface localisation of the SceA-GFP fusion protein on vegetative cells and spores was confirmed by Western blot, immunofluorescence microscopy and flow cytometry. The GFP fluorescence intensity from both vegetative cells and spores was measured and compared to a previously characterised surface display system using a peptidoglycan hydrolase anchor (Mbg). Results demonstrated comparable efficiency of SceA- and Mbg-mediated immobilisation on vegetative cells but a more efficient immobilisation on spores using the SceA anchor, suggesting SceA has greater potential for spore-based applications. The SceA protein was then applied to target BglS onto vegetative cells and spores, and the surface immobilisation was verified by the substantial whole-cell enzymatic activity and enhanced whole-spore enzymatic activity compared to vegetative cells. A dually active B. thuringiensis vegetative cell and spore display system could prove especially valuable for the development of regenerable and heat-stable biocatalysts that function under adverse environmental conditions, for example, an effective feed additive for improved digestion and nutrient absorption by livestock.

  17. The Synergistic Effect of High Pressure CO2 and Nisin on Inactivation of Bacillus subtilis Spores in Aqueous Solutions

    Science.gov (United States)

    Rao, Lei; Wang, Yongtao; Chen, Fang; Liao, Xiaojun

    2016-01-01

    The inactivation effects of high pressure CO2 + nisin (simultaneous treatment of HPCD and nisin, HPCD + nisin), HPCD→nisin (HPCD was followed by nisin), and nisin→HPCD (nisin was followed by HPCD) treatments on Bacillus subtilis spores in aqueous solutions were compared. The spores were treated by HPCD at 6.5 or 20 MPa, 84–86°C and 0–30 min, and the concentration of nisin was 0.02%. Treated spores were examined for the viability, the permeability of inner membrane (IM) using flow cytometry method and pyridine-2, 6-dicarboxylic acid (DPA) release, and structural damage by transmission electron microscopy. A synergistic effect of HPCD + nisin treatment on inactivation of the spores was found, and the inactivation efficiency of the spores was HPCD + nisin > HPCD→nisin or nisin→HPCD. Moreover, HPCD + nisin caused higher IM permeability and DPA release of the spores than HPCD. A possible action mode of nisin-enhanced inactivation of the spores was suggested as that HPCD firstly damaged the coat and cortex of spores, and nisin penetrated into and acted on the IM of spores, which increased the damage to the IM of spores, and resulted in higher inactivation of the spores. PMID:27708639

  18. Killing of Bacillus Megaterium Spores by X-rays at the Phosphorus K-edge

    Science.gov (United States)

    Richmond, Robert C.; Frigo, Sean P.; Ehret, Charles F.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    This study continues a progression of experiments on the radiation-induced killing of bacterial spores that began at the Argonne National Laboratory in 1957. A series of aliquots of Bacillus megaterium spores were prepared onto polycarbonate filters and irradiated with photons of 2159 eV compared to 2140 eV energy on the 2-IDB beamline at the Advanced Photon Source. Flux density was approximately 10(exp 18) photons/sec/sq mm. The phosphorous K-edge absorption spectrum in these spores was determined to peak at 2159 eV, wheras 2140 eV was determined to be outside that absorption spectrum. Spores on filters were irradiated at ambient conditions, and were either immediately plated for colony formation after irradiation, or were held for postirradiation exposure to oxygen prior to plating. Slopes of survival curves from the four conditions of irradiation, i.e., two photon energies each comparing immediate plating vs postirradiation holding, were used for quantitative determination of differences in rates of spore killing over a range of radiation doses. It was found that spores irradiated at the phosphorus K-edge were killed 20% more efficiently than when irradiated with 2140 eV photons, and this was true for both immediate plating and postirradiation holding in air. Postirradiation holding in air increased killing efficiency by about 12% for both photon energies compared to plating immediately after irradiation. The increase of killing efficiency with postirradiation holding is less than expected from earlier experiments using relatively low-flux X-rays, and raises the possibility of dose-mitigation by radical-radical recombination in the case of high-flux X-rays from the synchrotron.

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

    Energy Technology Data Exchange (ETDEWEB)

    Letant, S E; Kane, S R; Murphy, G A; Alfaro, T M; Hodges, L; Rose, L; Raber, E

    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 of 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.

  20. Comprehensive Assignment of Mass Spectral Signatures from Individual Bacillus atrophaeus Spores in Matrix-Free Bioaerosol Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, A; Pitesky, M; Steele, P; Tobias, H; Fergenson, D P; Horn, J; Russell, S C; Czerwieniec, G; Lebrilla, C; Gard, E E; Frank, M

    2004-10-22

    We have conducted studies to fully characterize the mass spectral signature of individual Bacillus atrophaeus, previously known as Bacillus subtilis var niger or Bacillus globigii, spores obtained in matrix-free bioaerosol mass spectrometry (BAMS). Mass spectra of spores grown in unlabeled, {sup 13}C-labeled and {sup 15}N-labeled growth media are used to determine the number of carbon and nitrogen atoms associated with each mass peak. To determine the parent ion structure associated with fragment ions present in the spore spectra, the mass-to-charge (m/z) fragmentation pattern of several chemical standards was obtained. Our results agree with prior assignments of dipicolinic acid, amino acids and calcium complex ions made in the spore mass spectra. Identity of several previously unidentified mass peaks, key to recognition of Bacillus spore by matrix-free BAMS, is revealed. Specifically, a set of fragment peaks in the negative polarity is shown to be consistent with the fragmentation pattern of purine nucleobase containing compounds. The identity of m/z=+74, a marker peak that helps discriminate Bacillus atrophaeus from Bacillus thuringiensis spores grown in rich medium, is surprisingly a non-description, viz. [N{sub 1}C{sub 4}H{sub 12}]{sup +}. A probable precursor molecule for the [N{sub 1}C{sub 4}H{sub 12}]{sup +} ion observed in spore spectra is trimethyl glycine ({sup +}N(CH{sub 3}){sub 3}CH{sub 2}COOH) that produces a m/z=74 peak in presence of dipicolinic acid.

  1. Film coating of seeds with Bacillus cereus RS87 spores for early plant growth enhancement.

    Science.gov (United States)

    Jetiyanon, Kanchalee; Wittaya-Areekul, Sakchai; Plianbangchang, Pinyupa

    2008-10-01

    The plant growth-promoting rhizobacterium Bacillus cereus RS87 was previously reported to promote plant growth in various crops in both greenhouse and field trials. To apply as a plant growth promoting agent with practical use, it is essential to ease the burden of routine preparation of a fresh suspension of strain RS87 in laboratory. The objectives of this study were to investigate the feasibility of film-coating seeds with B. cereus RS87 spores for early plant growth enhancement and to reveal the indoleacetic acid (IAA) production released from strain RS87. The experiment consisted of the following 5 treatments: nontreated seeds, water-soaked seeds, film-coated seeds, seeds soaked with vegetative cells of strain RS87, and film-coated seeds with strain RS87 spores. Three experiments were conducted separately to assess seed emergence, root length, and plant height. Results showed that both vegetative cells and spores of strain RS87 significantly promoted (P seed emergence, root length and plant height over the control treatments. The strain RS87 also produced IAA. In conclusion, the film coating of seeds with spores of B. cereus RS87 demonstrated early plant growth enhancement as well as seeds using their vegetative cells. IAA released from strain RS87 would be one of the mechanisms for plant growth enhancement.

  2. Bacillus subtilis spores on artificial meteorites survive hypervelocity atmospheric entry: implications for Lithopanspermia.

    Science.gov (United States)

    Fajardo-Cavazos, Patricia; Link, Lindsey; Melosh, H Jay; Nicholson, Wayne L

    2005-12-01

    An important but untested aspect of the lithopanspermia hypothesis is that microbes situated on or within meteorites could survive hypervelocity entry from space through Earth's atmosphere. The use of high-altitude sounding rockets to test this notion was explored. Granite samples permeated with spores of Bacillus subtilis strain WN511 were attached to the exterior telemetry module of a sounding rocket and launched from White Sands Missile Range, New Mexico into space, reaching maximum atmospheric entry velocity of 1.2 km/s. Maximum recorded temperature during the flight was measured at 145 degrees C. The surfaces of the post-flight granite samples were swabbed and tested for recovery and survival of WN511 spores, using genetic markers and the unique DNA fingerprint of WN511 as recovery criteria. Spore survivors were isolated at high frequency, ranging from 1.2% to 4.4% compared with ground controls, from all surfaces except the forward-facing surface. Sporulation-defective mutants were noted among the spaceflight survivors at high frequency (4%). These experiments constitute the first report of spore survival to hypervelocity atmospheric transit, and indicate that sounding rocket flights can be used to model the high-speed atmospheric entry of bacteria-laden artificial meteorites.

  3. Identification of the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available Immunoproteomics was used to screen the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R. The spore and vegetative proteins were separated by 2D gel electrophoresis and transferred to polyvinylidene difluoride membranes, and then western blotting was performed with rabbit immune serum against B.anthracis live spores. Immunogenic spots were cut and digested by trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed to identify the proteins. As a result, 11 and 45 immunogenic proteins were identified in the spores and vegetative cells, respectively; 26 of which have not been reported previously. To verify their immunogenicity, 12 of the identified proteins were selected to be expressed, and the immune sera from the mice vaccinated by the 12 expressed proteins, except BA0887, had a specific western blot band with the A16R whole cellular lytic proteins. Some of these immunogenic proteins might be used as novel vaccine candidates themselves or for enhancing the protective efficacy of a protective-antigen-based vaccine.

  4. Variable Lymphocyte Receptor Recognition of the Immunodominant Glycoprotein of Bacillus anthracis Spores

    Energy Technology Data Exchange (ETDEWEB)

    Kirchdoerfer, Robert N.; Herrin, Brantley R.; Han, Byung Woo; Turnbough, Jr., Charles L.; Cooper, Max D.; Wilson, Ian A. (SNU); (Scripps); (Emory); (UAB); (Emory Vaccine)

    2012-07-25

    Variable lymphocyte receptors (VLRs) are the adaptive immune receptors of jawless fish, which evolved adaptive immunity independent of other vertebrates. In lieu of the immunoglobulin fold-based T and B cell receptors, lymphocyte-like cells of jawless fish express VLRs (VLRA, VLRB, or VLRC) composed of leucine-rich repeats and are similar to toll-like receptors (TLRs) in structure, but antibodies (VLRB) and T cell receptors (VLRA and VLRC) in function. Here, we present the structural and biochemical characterization of VLR4, a VLRB, in complex with BclA, the immunodominant glycoprotein of Bacillus anthracis spores. Using a combination of crystallography, mutagenesis, and binding studies, we delineate the mode of antigen recognition and binding between VLR4 and BclA, examine commonalities in VLRB recognition of antigens, and demonstrate the potential of VLR4 as a diagnostic tool for the identification of B. anthracis spores.

  5. Disinfection and regrowth potential of bacillus subtilis spores by ozone, ultraviolet rays and gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hae Yeon; Lee, O Mi; Kim, Tae Hun; Lee, Myun Joo; Yu, Seung Ho [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2009-06-15

    Chlorination has been the most commonly adopted disinfection process for the treatment of drinking water. However, Cryptosporidium parvum oocysts and Giardia lamblia cysts were not treated effectively by the common chlorine-based disinfectants. Additionally the regrowth of pathogenic microorganisms is associated with hygienic and aesthetic problems for the consumers of drinking water. Study on alternative disinfection processes such as ozone, UV-C, VUV and gamma irradiation were conducted. Bacillus subtilis spores have been used as a surrogate microorganism for Cryptosporidium parvum oocysts and Giardia lamblia cyst. Inactivation efficiency by ozone was from 30% to 96% within the range of 5 min to 120 min exposures. Inactivation efficiencies by UV-C and VUV were 95.18%, 95.07% at 30 sec, respectively. Inactivation efficiency at gamma irradiation dose of 2 kGy was 99.4%. Microbial regrowths after ozone, UV-C, VUV and gamma irradiation disinfections were also evaluated for 4 days. Bacillus subtilis spores after ozone treatment for 120 min exposure at the rate of 1.68 mg {center_dot} min{sup -1} showed 96.02% disinfection efficiency and significant microbial regrowth. Bacillus subtilis spores after UV-C (99.25% disinfection efficiency) and VUV (99.67% disinfection efficiency) treatments for 5 min showed gradual regrowth. However, inactivation efficiency of gamma irradiation at dose of 1 kGy was 98.8% and the disinfected sample showed no microbial regrowth for 4 days. Therefore, gamma irradiation is the most effective process for the disinfection of pathogenic microorganisms such as oocysts of protozoan parasites among four disinfection process.

  6. Kinetics of Mn(II) oxidation by spores of the marine Bacillus sp. SG-1

    Science.gov (United States)

    Toyoda, Kazuhiro; Tebo, Bradley M.

    2016-09-01

    The kinetics of Mn(II) oxidation by spores of the marine Bacillus sp. SG-1 was measured under controlled conditions of the initial Mn(II) concentration, spore concentration, chemical speciation, pH, O2, and temperature. Mn(II) oxidation experiments were performed with spore concentrations ranging from 0.7 to 11 × 109 spores/L, a pH range from 5.8 to 8.1, temperatures between 4 and 58 °C, a range of dissolved oxygen from 2 to 270 μM, and initial Mn(II) concentrations from 1 to 200 μM. The Mn(II) oxidation rates were directly proportional to the spore concentrations over these ranges of concentration. The Mn(II) oxidation rate increased with increasing initial Mn(II) concentration to a critical concentration, as described by the Michaelis-Menten model (Km = ca. 3 μM). Whereas with starting Mn(II) concentrations above the critical concentration, the rate was almost constant in low ionic solution (I = 0.05, 0.08). At high ionic solution (I = 0.53, 0.68), the rate was inversely correlated with Mn(II) concentration. Increase in the Mn(II) oxidation rate with the dissolved oxygen concentration followed the Michaelis-Menten model (Km = 12-19 μM DO) in both a HEPES-buffered commercial drinking (soft) water and in artificial and natural seawater. Overall, our results suggest that the mass transport limitations of Mn(II) ions due to secondary Mn oxide products accumulating on the spores cause a significant decrease of the oxidation rate at higher initial Mn(II) concentration on a spore basis, as well as in more concentrated ionic solutions. The optimum pH for Mn(II) oxidation was approximately 7.0 in low ionic solutions (I = 0.08). The high rates at the alkaline side (pH > 7.5) may suggest a contribution by heterogeneous reactions on manganese bio-oxides. The effect of temperature on the Mn(II) oxidation rate was studied in three solutions (500 mM NaCl, ASW, NSW solutions). Thermal denaturation occurred at 58 °C and spore germination was evident at 40 °C in all three

  7. A genetic algorithm-Bayesian network approach for the analysis of metabolomics and spectroscopic data: application to the rapid identification of Bacillus spores and classification of Bacillus species

    OpenAIRE

    Goodacre Royston; Correa Elon

    2011-01-01

    Abstract Background The rapid identification of Bacillus spores and bacterial identification are paramount because of their implications in food poisoning, pathogenesis and their use as potential biowarfare agents. Many automated analytical techniques such as Curie-point pyrolysis mass spectrometry (Py-MS) have been used to identify bacterial spores giving use to large amounts of analytical data. This high number of features makes interpretation of the data extremely difficult We analysed Py-...

  8. Detection of Bacillus anthracis Spores Using Peptide Functionalized SERS-Active Substrates

    Directory of Open Access Journals (Sweden)

    Atanu Sengupta

    2012-01-01

    Full Text Available The need for portable technologies that can rapidly identify biological warfare agents (BWAs in the field remains an international priority as expressed at the 2011 Biological Weapons Convention. In recent years, the ability of surface-enhanced Raman spectroscopy (SERS to rapidly detect various BWAs at very low concentrations has been demonstrated. However, in the specific case of Bacillus anthracis, differentiation at the species level is required since other bacilli are common in the environment, representing potential false-positive responses. To overcome this limitation, we describe the use of a peptide attached to the SERS-active metal that selectively binds Bacillus anthracis-Sterne as the target analyte. Using this approach, 109  B. anthracis-Sterne spores/mL produced an intense dipicolinic acid spectrum upon the addition of acetic acid, while the same concentration and treatment of B. cereus and B. subtilis did not.

  9. Online monitoring of Escherichia coli and Bacillus thuringiensis spore inactivation after advanced oxidation treatment.

    Science.gov (United States)

    Sherchan, Samendra P; Snyder, Shane A; Gerba, Charles P; Pepper, Ian L

    2014-01-01

    Various studies have shown that advanced oxidation processes (AOPs) such as UV light in combination with hydrogen peroxide is an efficient process for the removal of a large variety of emerging contaminants including microorganisms. The mechanism of destruction in the presence of hydrogen peroxide (H2O2) is the enhanced formation of hydroxyl (·OH) radicals, which have a high oxidation potential. The goal of this study was to utilize in-line advanced oxidation to inactivate microbes, and document the inactivation via an in-line, real-time sensor. Escherichia coli cells and Bacillus thuringiensis spores were exposed to UV/H2O2 treatment in DI water, and the online sensor BioSentry(®) was evaluated for its potential to monitor inactivation in real-time. B. thuringiensis was selected as a non-pathogenic surrogate for B. anthracis, the causative agent of anthrax and a proven biological weapon. UV radiation and UV/H2O2 exposure resulted in a >6 log10 reduction of the viable culturable counts of E. coli vegetative cells, and a 3 log10 reduction of B. thuringiensis spores. Scanning electron microscopy of the treated samples revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the morphology of the B. thuringiensis spores. Following AOP exposure, the BioSentry sensor showed an increase in the categories of unknown, rod and spores counts, but overall, did not correspond well with viable count assays. Data from this study show that advanced oxidation processes effectively inactivate E. coli vegetative cells, but not B. thuringiensis spores, which were more resistant to AOP. Further, the BioSentry in-line sensor was not successful in documenting destruction of the microbial cells in real-time.

  10. Rapid Detection of Viable Bacillus anthracis Spores in Environmental Samples by Using Engineered Reporter Phages

    Science.gov (United States)

    Sharp, Natasha J.; Molineux, Ian J.; Page, Martin A.

    2016-01-01

    Bacillus anthracis, the causative agent of anthrax, was utilized as a bioterrorism agent in 2001 when spores were distributed via the U.S. postal system. In responding to this event, the Federal Bureau of Investigation used traditional bacterial culture viability assays to ascertain the extent of contamination of the postal facilities within 24 to 48 h of environmental sample acquisition. Here, we describe a low-complexity, second-generation reporter phage assay for the rapid detection of viable B. anthracis spores in environmental samples. The assay uses an engineered B. anthracis reporter phage (Wβ::luxAB-2) which transduces bioluminescence to infected cells. To facilitate low-level environmental detection and maximize the signal response, expression of luxAB in an earlier version of the reporter phage (Wβ::luxAB-1) was optimized. These alterations prolonged signal kinetics, increased light output, and improved assay sensitivity. Using Wβ::luxAB-2, detection of B. anthracis spores was 1 CFU in 8 h from pure cultures and as low as 10 CFU/g in sterile soil but increased to 105 CFU/g in unprocessed soil due to an unstable signal and the presence of competing bacteria. Inclusion of semiselective medium, mediated by a phage-expressed antibiotic resistance gene, maintained signal stability and enabled the detection of 104 CFU/g in 6 h. The assay does not require spore extraction and relies on the phage infecting germinating cells directly in the soil sample. This reporter phage displays promise for the rapid detection of low levels of spores on clean surfaces and also in grossly contaminated environmental samples from complex matrices such as soils. PMID:26873316

  11. Rapid Detection of Viable Bacillus anthracis Spores in Environmental Samples by Using Engineered Reporter Phages.

    Science.gov (United States)

    Sharp, Natasha J; Molineux, Ian J; Page, Martin A; Schofield, David A

    2016-04-01

    Bacillus anthracis, the causative agent of anthrax, was utilized as a bioterrorism agent in 2001 when spores were distributed via the U.S. postal system. In responding to this event, the Federal Bureau of Investigation used traditional bacterial culture viability assays to ascertain the extent of contamination of the postal facilities within 24 to 48 h of environmental sample acquisition. Here, we describe a low-complexity, second-generation reporter phage assay for the rapid detection of viableB. anthracis spores in environmental samples. The assay uses an engineered B. anthracis reporter phage (Wβ::luxAB-2) which transduces bioluminescence to infected cells. To facilitate low-level environmental detection and maximize the signal response, expression of luxABin an earlier version of the reporter phage (Wβ::luxAB-1) was optimized. These alterations prolonged signal kinetics, increased light output, and improved assay sensitivity. Using Wβ::luxAB-2, detection of B. anthracis spores was 1 CFU in 8 h from pure cultures and as low as 10 CFU/g in sterile soil but increased to 10(5)CFU/g in unprocessed soil due to an unstable signal and the presence of competing bacteria. Inclusion of semiselective medium, mediated by a phage-expressed antibiotic resistance gene, maintained signal stability and enabled the detection of 10(4)CFU/g in 6 h. The assay does not require spore extraction and relies on the phage infecting germinating cells directly in the soil sample. This reporter phage displays promise for the rapid detection of low levels of spores on clean surfaces and also in grossly contaminated environmental samples from complex matrices such as soils.

  12. Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination.

    Science.gov (United States)

    Korza, George; Setlow, Barbara; Rao, Lei; Li, Qiao; Setlow, Peter

    2016-12-15

    rRNAs of dormant spores of Bacillus subtilis were >95% degraded during extended incubation at 50°C, as reported previously (E. Segev, Y. Smith, and S. Ben-Yehuda, Cell 148:139-114, 2012, doi:http://dx.doi.org/10.1016/j.cell.2011.11.059), and this was also true of spores of Bacillus megaterium Incubation of spores of these two species for ∼20 h at 75 to 80°C also resulted in the degradation of all or the great majority of the 23S and 16S rRNAs, although this rRNA degradation was slower than nonenzymatic hydrolysis of purified rRNAs at these temperatures. This rRNA degradation at high temperature generated almost exclusively oligonucleotides with minimal levels of mononucleotides. RNase Y, suggested to be involved in rRNA hydrolysis during B. subtilis spore incubation at 50°C, did not play a role in B. subtilis spore rRNA breakdown at 80°C. Twenty hours of incubation of Bacillus spores at 70°C also decreased the already minimal levels of ATP in dormant spores 10- to 30-fold, to ≤0.01% of the total free adenine nucleotide levels. Spores depleted of rRNA were viable and germinated relatively normally, often even faster than starting spores. Their return to vegetative growth was also similar to that of untreated spores for B. megaterium spores and slower for heat-treated B. subtilis spores; accumulation of rRNA took place only after completion of spore germination. These findings thus strongly suggest that protein synthesis is not essential for Bacillus spore germination.IMPORTANCE A recent report (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:3486-3495, 2015, doi:http://dx.doi.org/10.1016/j.molcel.2014.12.019) suggested that protein synthesis is essential for early steps in the germination of dormant spores of Bacillus subtilis If true, this would be a paradigm shift in our understanding of spore germination. We now show that essentially all of the rRNA can be eliminated from spores of Bacillus megaterium or B. subtilis, and these

  13. Modelling the effect of sub(lethal) heat treatment of Bacillus subtilis spores on germination rate and outgrowth to exponentially growing vegetative cells

    NARCIS (Netherlands)

    Smelt, J.P.P.M.; Bos, A.P.; Kort, R.; Brul, S.

    2008-01-01

    Spores of Bacillus subtilis were subjected to relatively mild heat treatments in distilled water and properties of these spores were studied. These spores had lost all or part of their dipicolinic acid (DPA) depending on the severity of the heat treatment. Even after relatively mild heat treatments

  14. Micro-Etched Platforms for Thermal Inactivation of Bacillus Anthracis and Bacillus Thuringiensis Spores

    Science.gov (United States)

    2008-03-01

    furnace. A silicon nitride base provided a uniformly heated surface which ensured a uniform temperature exposure for the entire platform surface ...holding spores was wet chemical etching. This procedure entailed several steps in order to prepare the glass surface to be etched. Cleanliness of the...inspected by light microscopy at multiple intervals for surface cleanliness . 40 Figure 17. 400x magnification of Cover slip after the surface has

  15. Comparison of four commercial DNA extraction kits for the recovery of Bacillus spp. spore DNA from spiked powder samples.

    Science.gov (United States)

    Mölsä, Markos; Kalin-Mänttäri, Laura; Tonteri, Elina; Hemmilä, Heidi; Nikkari, Simo

    2016-09-01

    Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×10(1)CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×10(3)CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity.

  16. Direct investigation of viscosity of an atypical inner membrane of Bacillus spores: a molecular rotor/FLIM study.

    Science.gov (United States)

    Loison, Pauline; Hosny, Neveen A; Gervais, Patrick; Champion, Dominique; Kuimova, Marina K; Perrier-Cornet, Jean-Marie

    2013-11-01

    We utilize the fluorescent molecular rotor Bodipy-C12 to investigate the viscoelastic properties of hydrophobic layers of bacterial spores Bacillus subtilis. The molecular rotor shows a marked increase in fluorescence lifetime, from 0.3 to 4ns, upon viscosity increase from 1 to 1500cP and can be incorporated into the hydrophobic layers within the spores from dormant state through to germination. We use fluorescence lifetime imaging microscopy to visualize the viscosity inside different compartments of the bacterial spore in order to investigate the inner membrane and relate its compaction to the extreme resistance observed during exposure of spores to toxic chemicals. We demonstrate that the bacterial spores possess an inner membrane that is characterized by a very high viscosity, exceeding 1000cP, where the lipid bilayer is likely in a gel state. We also show that this membrane evolves during germination to reach a viscosity value close to that of a vegetative cell membrane, ca. 600cP. The present study demonstrates quantitative imaging of the microscopic viscosity in hydrophobic layers of bacterial spores Bacillus subtilis and shows the potential for further investigation of spore membranes under environmental stress.

  17. Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells.

    Science.gov (United States)

    Walper, Scott A; Anderson, George P; Brozozog Lee, P Audrey; Glaven, Richard H; Liu, Jinny L; Bernstein, Rachel D; Zabetakis, Dan; Johnson, Linwood; Czarnecki, Jill M; Goldman, Ellen R

    2012-01-01

    Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.

  18. ON THE USE OF FROTH FLOTATION IN THE RECOVERY OF Bacillus sphaericus SPORES

    Directory of Open Access Journals (Sweden)

    E.M. RIOS

    1997-06-01

    Full Text Available Abstract - The recovery of Bacillus sphaericus strain 2362 spores from fermented medium by batch flotation was tested under different conditions. Flotation kinetic studies were performed at 800 rpm and 3 l air/min. The pH values were adjusted at the following set of values: 5.0, 7.0 and 9.0. The results showed that the spore removal rate is influenced by the pH value. At pH equal to 5.0 we observe an adverse effect on the spore concentrate obtention. In this situation the maximum value of the concentration factor was 1.4 when the recuperation percentage was 99%. At pH equal to 7.0 the concentration factor reached the highest value, 7.0, but the recuperation percentage stayed around 96%. Field experiments with the floated material demonstrated that its larvicide activity was sufficient to keep a Culex quinquefasciatus larvae population under control at in a breeding site, during 3 months with 2 applications

  19. Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells.

    Directory of Open Access Journals (Sweden)

    Scott A Walper

    Full Text Available Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.

  20. Bacillus spores as building blocks for stimuli-responsive materials and nanogenerators

    Science.gov (United States)

    Chen, Xi; Mahadevan, L.; Driks, Adam; Sahin, Ozgur

    2014-02-01

    Materials that respond mechanically to external chemical stimuli have applications in biomedical devices, adaptive architectural systems, robotics and energy harvesting. Inspired by biological systems, stimuli-responsive materials have been created that can oscillate, transport fluid, provide homeostasis and undergo complex changes in shape. However, the effectiveness of synthetic stimuli-responsive materials in generating work is limited when compared with mechanical actuators. Here, we show that the mechanical response of Bacillus spores to water gradients exhibits an energy density of more than 10 MJ m-3, which is two orders of magnitude higher than synthetic water-responsive materials. We also identified mutations that can approximately double the energy density of the spores and found that they can self-assemble into dense, submicrometre-thick monolayers on substrates such as silicon microcantilevers and elastomer sheets, creating bio-hybrid hygromorph actuators. To illustrate the potential applications of the spores, we used them to build an energy-harvesting device that can remotely generate electrical power from an evaporating body of water.

  1. Optimization of a sample processing protocol for recovery of Bacillus anthracis spores from soil

    Science.gov (United States)

    Silvestri, Erin E.; Feldhake, David; Griffin, Dale; Lisle, John T.; Nichols, Tonya L.; Shah, Sanjiv; Pemberton, A; Schaefer III, Frank W

    2016-01-01

    Following a release of Bacillus anthracis spores into the environment, there is a potential for lasting environmental contamination in soils. There is a need for detection protocols for B. anthracis in environmental matrices. However, identification of B. anthracis within a soil is a difficult task. Processing soil samples helps to remove debris, chemical components, and biological impurities that can interfere with microbiological detection. This study aimed to optimize a previously used indirect processing protocol, which included a series of washing and centrifugation steps. Optimization of the protocol included: identifying an ideal extraction diluent, variation in the number of wash steps, variation in the initial centrifugation speed, sonication and shaking mechanisms. The optimized protocol was demonstrated at two laboratories in order to evaluate the recovery of spores from loamy and sandy soils. The new protocol demonstrated an improved limit of detection for loamy and sandy soils over the non-optimized protocol with an approximate matrix limit of detection at 14 spores/g of soil. There were no significant differences overall between the two laboratories for either soil type, suggesting that the processing protocol will be robust enough to use at multiple laboratories while achieving comparable recoveries.

  2. Investigating synergism during sequential inactivation of Bacillus subtilis spores with several disinfectants.

    Science.gov (United States)

    Cho, Min; Kim, Jae-Hong; Yoon, Jeyong

    2006-08-01

    The sequential application of ozone, chlorine dioxide, or UV followed by free chlorine was performed to investigate the synergistic inactivation of Bacillus subtilis spores. The greatest synergism was observed when chlorine dioxide was used as a primary disinfectant followed by secondary disinfection with free chlorine. A lesser synergistic effect was observed when ozone was used as the primary disinfectant, but no synergism was observed when UV was used as the primary disinfectant. When free chlorine was used as the primary disinfectant (i.e., sequential application in the reverse order), the synergistic effect was shown only when chlorine dioxide was applied as the secondary disinfectant. The synergistic effect observed could be related to damage to the spore coat during primary disinfection, suggested by the loss of proteins from spores during disinfectant treatment. The greatest synergism observed by the chlorine dioxide/free chlorine pair suggested that common reaction sites might exist for these disinfectants. The concept of percent synergistic effect was introduced to quantitatively compare the extent of synergistic effects in the sequential disinfection processes.

  3. Achieving consistent multiple daily low-dose Bacillus anthracis spore inhalation exposures in the rabbit model

    Directory of Open Access Journals (Sweden)

    Roy E Barnewall

    2012-06-01

    Full Text Available Repeated low-level exposures to Bacillus anthracis could occur before or after the remediation of an environmental release. This is especially true for persistent agents such as Bacillus anthracis spores, the causative agent of anthrax. Studies were conducted to examine aerosol methods needed for consistent daily low aerosol concentrations to deliver a low-dose (less than 106 colony forming units (CFU of B. anthracis spores and included a pilot feasibility characterization study, acute exposure study, and a multiple fifteen day exposure study. This manuscript focuses on the state-of-the-science aerosol methodologies used to generate and aerosolize consistent daily low aerosol concentrations and resultant low inhalation doses. The pilot feasibility characterization study determined that the aerosol system was consistent and capable of producing very low aerosol concentrations. In the acute, single day exposure experiment, targeted inhaled doses of 1 x 102, 1 x 103, 1 x 104, and 1 x 105 CFU were used. In the multiple daily exposure experiment, rabbits were exposed multiple days to targeted inhaled doses of 1 x 102, 1 x 103, and 1 x 104 CFU. In all studies, targeted inhaled doses remained fairly consistent from rabbit to rabbit and day to day. The aerosol system produced aerosolized spores within the optimal mass median aerodynamic diameter particle size range to reach deep lung alveoli. Consistency of the inhaled dose was aided by monitoring and recording respiratory parameters during the exposure with real-time plethysmography. Overall, the presented results show that the animal aerosol system was stable and highly reproducible between different studies and multiple exposure days.

  4. Decontamination of Bacillus subtilis var. niger spores on selected surfaces by chlorine dioxide gas*

    Science.gov (United States)

    Li, Yan-ju; Zhu, Neng; Jia, Hai-quan; Wu, Jin-hui; Yi, Ying; Qi, Jian-cheng

    2012-01-01

    Objective: Chlorine dioxide (CD) gas has been used as a fumigant in the disinfection of biosafety laboratories. In this study, some experiments were conducted to assess the inactivation of spores inoculated on six materials [stainless steel (SS), painted steel (PS), polyvinyl chlorid (PVC), polyurethane (PU), glass (GS), and cotton cloth (CC)] by CD gas. The main aims of the study were to determine the sporicidal efficacy of CD gas and the effect of prehumidification before decontamination on sporicidal efficacy. Methods: Material coupons (1.2 cm diameter of SS, PS, and PU; 1.0 cm×1.0 cm for PVC, GS, and CC) were contaminated with 10 μl of Bacillus subtilis var. niger (ATCC 9372) spore suspension in mixed organic burden and then dried in a biosafety cabinet for 12 h. The spores were recovered by soaking the coupons in 5 ml of extraction liquid for 1 h and then vortexing the liquid for 1 min. Results: The log reductions in spore numbers on inoculated test materials exposed to CD gas [0.080% (volume ratio, v/v) for 3 h] were in the range of from 1.80 to 6.64. Statistically significant differences were found in decontamination efficacies on test material coupons of SS, PS, PU, and CC between with and without a 1-h prehumidification treatment. With the extraction method, there were no statistically significant differences in the recovery ratios between the porous and non-porous materials. Conclusions: The results reported from this study could provide information for developing decontamination technology based on CD gas for targeting surface microbial contamination. PMID:22467366

  5. Decontamination of Bacillus subtilis var.niger spores on selected surfaces by chlorine dioxide gas

    Institute of Scientific and Technical Information of China (English)

    Yan-ju LI; Neng ZHU; Hai-quan JIA; Jin-hui WU; Ying YI; Jian-cheng QI

    2012-01-01

    Objective:Chlorine dioxide (CD) gas has been used as a fumigant in the disinfection of biosafety laboratories.In this study,some experiments were conducted to assess the inactivation of spores inoculated on six materials [stainless steel (SS),painted steel (PS),polyvinyl chlorid (PVC),polyurethane (PU),glass (GS),and cotton cloth (CC)] by CD gas.The main aims of the study were to determine the sporicidal efficacy of CD gas and the effect of prehumidification before decontamination on sporicidal efficacy.Methods:Material coupons (1.2 cm diameter of SS,PS,and PU; 1.0 cm×1.0 cm for PVC,GS,and CC) were contaminated with 10 μl of Bacillus subtilis var.niger(ATCC 9372) spore suspension in mixed organic burden and then dried in a biosafety cabinet for 12 h.The spores were recovered by soaking the coupons in 5 ml of extraction liquid for 1 h and then vortexing the liquid for 1 min.Results:The log reductions in spore numbers on inoculated test materials exposed to CD gas [0.080% (volume ratio,v/v) for 3 h]were in the range of from 1.80 to 6.64.Statistically significant differences were found in decontamination efficacies on test material coupons of SS,PS,PU,and CC between with and without a 1-h prehumidification treatment.With the extraction method,there were no statistically significant differences in the recovery ratios between the porous and non-porous materials.Conclusions:The results reported from this study could provide information for developing decontamination technology based on CD gas for targeting surface microbial contamination.

  6. Adsorption of β-galactosidase of Alicyclobacillus acidocaldarius on wild type and mutants spores of Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Sirec Teja

    2012-08-01

    Full Text Available Abstract Background The Bacillus subtilis spore has long been used as a surface display system with potential applications in a variety of fields ranging from mucosal vaccine delivery, bioremediation and biocatalyst development. More recently, a non-recombinant approach of spore display has been proposed and heterologous proteins adsorbed on the spore surface. We used the well-characterized β-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius as a model to study enzyme adsorption, to analyze whether and how spore-adsorption affects the properties of the enzyme and to improve the efficiency of the process. Results We report that purified β-galactosidase molecules were adsorbed to purified spores of a wild type strain of B. subtilis retaining ca. 50% of their enzymatic activity. Optimal pH and temperature of the enzyme were not altered by the presence of the spore, that protected the adsorbed β-galactosidase from exposure to acidic pH conditions. A collection of mutant strains of B. subtilis lacking a single or several spore coat proteins was compared to the isogenic parental strain for the adsorption efficiency. Mutants with an altered outermost spore layer (crust were able to adsorb 60-80% of the enzyme, while mutants with a severely altered or totally lacking outer coat adsorbed 100% of the β-galactosidase molecules present in the adsorption reaction. Conclusion Our results indicate that the spore surface structures, the crust and the outer coat layer, have an negative effect on the adhesion of the β-galactosidase. Electrostatic forces, previously suggested as main determinants of spore adsorption, do not seem to play an essential role in the spore-β-galactosidase interaction. The analysis of mutants with altered spore surface has shown that the process of spore adsorption can be improved and has suggested that such improvement has to be based on a better understanding of the spore surface structure

  7. Growth/no growth models for heat-treated psychrotrophic Bacillus cereus spores under cold storage.

    Science.gov (United States)

    Daelman, Jeff; Vermeulen, An; Willemyns, Tine; Ongenaert, Rebecca; Jacxsens, Liesbeth; Uyttendaele, Mieke; Devlieghere, Frank

    2013-01-15

    The microbiological safety of refrigerated and processed foods of extended durability (REPFED) is linked to spore-forming pathogens, more specifically Clostridium botulinum and Bacillus cereus. In this study two sets of growth/no growth (GNG) models are presented for the spores of two B. cereus strains. The models incorporate both product (water activity (a(w)) and pH) and process parameters (pasteurization value at 90 °C (P(90)) or heating temperature). The first model evaluates the effect of four different P(90)-values (P(90)=0, 4, 7 or 10 min, all applied at 90 °C) on the germination and subsequent growth of B. cereus spores under different conditions of pH and a(w) at 10 °C. These models show that a heat treatment not only increases the time to growth (TTG), but also significantly increases the minimal a(w) and pH necessary for germination and subsequent growth: e.g. at a(w) 0.995 and without heat treatment (P(90)=0), strain FF355 B. cereus spores were predicted to germinate and grow at pH 5.3. With a P(90) of 10 min, the minimal pH increased to 5.7. The second set of models for B. cereus spores compares the effect of three heat treatments with the same P(90)-value (10 min) but applied at different temperatures (85, 87 and 90 °C), on the germination and subsequent growth at 10 °C. The second model shows that lower heating temperatures (85 and 87 °C) had less effect on the TTG and minimal a(w) and pH than a higher temperature (90 °C). Finally, the first set of models was validated in broth using spores of seven psychrotrophic B. cereus strains, to evaluate the effect of strain variability on the model predictions. The results of the validation (% growth) were compared to the predicted growth probability. The results showed that the models were prone to fail-dangerous results (i.e. predicting no growth when growth was observed: 17%-34%). Using a very low threshold for growth (0.1% predicted chance of growth was considered to be complete growth), the models

  8. Early Warning of Biological Threats via Surface-Enhanced Raman Spectroscopy: A Case Study of Bacillus Spores

    Directory of Open Access Journals (Sweden)

    Antonia Lai

    2016-12-01

    Full Text Available A study on the application of surface-enhanced Raman spectroscopy (SERS in detecting biological threats is here reported. Simulants of deadly Bacillus anthracis endospores were used. This study proposes an automated device where SERS is used as a fast, pre-alarm technique of a two-stage sensor equipped with a real-time polymerase chain reaction (PCR. In order to check the potentialities of SERS in terms of sensitivity and specificity for on-site, real-time, automatic detection and identification of biological agents, two strains of genetically and harmless closely B. anthracis-related spores, Bacillus thuringiensis and Bacillus atrophaeus, were used as simulants. In order to assure the selectivity of the SERS substrate against B. thuringiensis spores, the substrate was functionalized by specific peptides. The obtained SERS measurements are classified as positive or negative hits by applying a special data evaluation based on the Euclidian distance between each spectrum and a reference spectrum of blank measurement. Principal component analysis (PCA was applied for discriminating between different strains representing dangerous and harmless spores. The results show that the SERS sensor is capable of detecting a few tenths of spores in a few minutes, and is particularly sensitive and fast for this purpose. Post-process analysis of the spectra allowed for discrimination between the contaminated and uncontaminated SERS sensors and even between different strains of spores, although not as clearly. For this purpose, the use of a non-functionalized SERS substrate is suggested.

  9. On-chip Detection of Rolling Circle Amplified DNA Molecules from Bacillus Globigii spores and Vibrio Cholerae

    DEFF Research Database (Denmark)

    Østerberg, Frederik Westergaard; Rizzi, Giovanni; Donolato, Marco

    2014-01-01

    , which makes the setup very compact. Limits of detection down to 500 Bacillus globigii spores and 2 pM of Vibrio cholerae are demonstrated, which are on the same order of magnitude or lower than those achieved previously using a commercial macro-scale AC susceptometer. The chipbased readout...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-06

    Rapid, cost-effective bacterial detection systems are needed to respond to potential biothreat events. Here we report the use of smartphone-based microscopy in combination with a simple microfluidic incubation device to detect 5000 Bacillus anthracis spores in 3 hours. This field-deployable approach is compatible with real-time PCR for secondary confirmation.

  11. 40 CFR 180.1076 - Viable spores of the microorganism Bacillus popilliae; exemption from the requirement of a...

    Science.gov (United States)

    2010-07-01

    ... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1076 Viable spores of the microorganism... have the following specifications: (1) The microorganism shall be an authentic strain of Bacillus... popilliae shall be produced by an extraction process from diseased Japanese beetles, and may contain a...

  12. Pilot-scale crossflow-microfiltration and pasturization to remove spores of Bacillus anthracis (Sterne) from milk

    Science.gov (United States)

    HTST pasteurization of milk is generally ineffective against spore-forming bacteria such as Bacillus anthracis (BA) but is lethal to its vegetative cells. Crossflow microfiltration (MF), using ceramic membranes with a pore diameter of 1.4 um, has been shown to physically remove somatic cells, vegeta...

  13. On the origin of heterogeneity in (preservation) resistance of Bacillus spores: input for a 'systems' analysis approach of bacterial spore outgrowth.

    Science.gov (United States)

    Hornstra, Luc M; Ter Beek, Alex; Smelt, Jan P; Kallemeijn, Wouter W; Brul, Stanley

    2009-08-31

    Bacterial spores are the ultimate (stress) 'survival capsules'. They allow strains from the Bacillus and Clostridium species to survive harsh environmental conditions. In addition to the decision to enter sporulation the decision to do the reverse (germinate) is also a decisive event after which there is no return. Generally it is observed that the behaviour of spores towards the environment is not homogeneous. In fact in many cases it is even quite heterogeneous, certainly upon subjecting the spores to a thermal stress treatment. Genome information coupled to high resolution single-cell analysis techniques allow us currently to analyse signalling events of individual cells. In the area of food preservation the next challenge is to couple the newly acquired mechanistic data to the physiologically observed heterogeneity in spore behaviour. The current paper will introduce the background of physiological heterogeneity while discussing the molecular processes that likely contribute to the observed heterogeneity in outgrowth. The discussion is set in the framework of contemporary and future needs for single-cell data integration in order to enhance the mechanistic basis of food preservation and spoilage models targeting bacterial spores.

  14. Amperometric Detection of Bacillus anthracis Spores: A Portable, Low-Cost Approach to the ELISA

    Directory of Open Access Journals (Sweden)

    Gabriel D. Peckham

    2013-01-01

    Full Text Available Antibody-based detection assays are generally robust, a desirable characteristic for in-the-field use. However, to quantify the colorimetric or fluorescent signal, these assays require expensive and fragile instruments which are ill-suited to in-the-field use. Lateral flow devices (LFDs circumvent these barriers to portability but suffer from poor sensitivity and subjective interpretation. Here, an antibody-based method for detecting Bacillus anthracis spores via amperometric signal generation is compared to ELISA and LFDs. This amperometric immunoassay uses antibody conjugated to magnetic beads and glucose oxidase (GOX along with the electron mediator 2, 6-dichlorophenolindophenol (DCPIP for production of a measurable current from a 0.4 V bias voltage. With similar sensitivity to ELISA, the assay can be completed in about 75 minutes while being completely powered and operated from a laptop computer. Immunoassay amperometry holds promise for bringing low-cost, quantitative detection of hazardous agents to the field.

  15. Phage-based magnetostrictive-acoustic microbiosensors for detecting bacillus anthracis spores

    Science.gov (United States)

    Wan, J.; Yang, H.; Lakshmanan, R. S.; Guntupalli, R.; Huang, S.; Hu, J.; Petrenko, V. A.; Chin, B. A.

    2006-05-01

    Magnetostrictive particles (MSPs) as biosensor platform have been developed recently. The principle of MSPs as sensor platform is the same as that of other acoustic wave devices, such as quartz crystal microbalance. In this paper, the fabrication, characterization and performance of phage-based MSP biosensors for detecting Bacillus anthracis spores are reported. A commercially available magnetostrictive alloy was utilized to fabricate the sensor platform. The phage was immobilized onto the MSPs using physical adsorption technology. The following performance of the phage-based MSP sensors will be presented: sensitivity, response time, longevity, specificity and binding efficacy. The performance of the sensors at static and dynamic conditions was characterized. The experimental results are confirmed by microscopy photographs. The excellent performance including high sensitivity and rapid response is demonstrated. More importantly, it is experimentally found that the phage-based MSP sensors have a much better longevity than antibody-based sensors.

  16. 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

    Science.gov (United States)

    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.

  17. Infrared decontamination of oregano: effects on Bacillus cereus spores, water activity, color, and volatile compounds.

    Science.gov (United States)

    Eliasson, Lovisa; Libander, Patrik; Lövenklev, Maria; Isaksson, Sven; Ahrné, Lilia

    2014-12-01

    Infrared (IR) heating, a novel technology for decontaminating oregano, was evaluated by investigating the reduction of inoculated Bacillus cereus spores and the effect on water activity (a(w)), color, and headspace volatile compounds after exposure to IR treatment. Conditioned oregano (a(w) 0.88) was IR-treated in a closed heating unit at 90 and 100 °C for holding times of 2 and 10 min, respectively. The most successful reduction in B. cereus spore numbers (5.6 log units) was achieved after a holding time of 10 min at 90 °C, while treatment at 100 °C for the same time resulted in a lower reduction efficiency (4.7 log units). The lower reduction at 100 °C was probably due to a reduced aw (aw 0.76) during IR treatment or possibly to the alteration or loss of volatile compounds possessing antimicrobial properties. The green color of oregano was only slightly affected, while the composition of volatile compounds was clearly altered by IR heating. However, two of the key aroma compounds, carvacrol and thymol, were only slightly affected, compared to the effect on the other studied compounds, indicating that the typical oregano aroma can likely be preserved. In conclusion, IR heating shows potential for the successful decontamination of oregano without severe alteration of its color or the key aroma compounds, carvacrol and thymol.

  18. Decrease in spermidine content during logarithmic phase of cell growth delays spore formation of Bacillus subtilis.

    Science.gov (United States)

    Ishii, I; Takada, H; Terao, K; Kakegawa, T; Igarashi, K; Hirose, S

    1994-11-01

    Bacillus subtilis 168M contained a large amount of spermidine during the logarithmic phase of growth, but the amount decreased drastically during the stationary phase. The extracts, prepared from B. subtilis cells harvested in the logarithmic phase, contained activity of arginine decarboxylase (ADC) rather than the activity of ornithine decarboxylase. In the presence of alpha-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of ADC, the amount of spermidine in B. subtilis during the logarithmic phase decreased to about 25% of the control cells. Under these conditions, spore formation of B. subtilis 168M delayed greatly without significant inhibition of cell growth. The decrease in spermidine content in the logarithmic phase rather than in the stationary phase was involved in the delay of sporulation. Electron microscopy of cells at 24 hrs. of culture confirmed the delay of spore formation by the decrease of spermidine content. Furthermore, the delay of sporulation was negated by the addition of spermidine. These data suggest that a large amount of spermidine existing during the logarithmic phase plays an important role in the sporulation of B. subtilis.

  19. Detection of agar, by analysis of sugar markers, associated with Bacillus anthracis spores, after culture.

    Science.gov (United States)

    Wunschel, David S; Colburn, Heather A; Fox, Alvin; Fox, Karen F; Harley, William M; Wahl, Jon H; Wahl, Karen L

    2008-08-01

    Detection of small quantities of agar associated with spores of Bacillus anthracis could provide key information regarding its source or growth characteristics. Agar, widely used in growth of bacteria on solid surfaces, consists primarily of repeating polysaccharide units of 3,6-anhydro-l-galactose (AGal) and galactose (Gal) with sulfated and O-methylated galactoses present as minor constituents. Two variants of the alditol acetate procedure were evaluated for detection of potential agar markers associated with spores. The first method employed a reductive hydrolysis step, to stabilize labile anhydrogalactose, by converting to anhydrogalactitol. The second eliminated the reductive hydrolysis step simplifying the procedure. Anhydrogalactitol, derived from agar, was detected using both derivatization methods followed by gas chromatography-mass spectrometry (GC-MS) analysis. However, challenges with artifactual background (reductive hydrolysis) or marker destruction (hydrolysis) respectively lead to the use of an alternative agar marker. A minor agar component, 6-O-methyl galactose (6-O-M gal), was readily detected in agar-grown but not broth-grown bacteria. Detection was optimized by the use of gas chromatography-tandem mass spectrometry (GC-MS-MS). With appropriate choice of sugar marker and analytical procedure, detection of sugar markers for agar has considerable potential in microbial forensics.

  20. Response of Bacillus subtilis spores to dehydration and UV irradiation at extremely low temperatures.

    Science.gov (United States)

    Dose, K; Klein, A

    1996-02-01

    Spores of Bacillus subtilis have been exposed to the conditions of extreme dehydration (argon/silica gel; simulated space vacuum) for up to 12 weeks at 298 K and 80 K in the dark. The inactivation has been correlated with the production of DNA-double strand-breaks. The temperature-dependence of the rate constants for inactivation or production of DNA-double strand-breaks is surprisingly low. Controls kept in the frozen state at 250 K for the same period of time showed no sign of deterioration. In another series of experiments the spores have been UV irradiated (253.7 nm) at 298 K, 200 K and 80 K after exposure to dehydrating conditions for 3 days. Fluence-effect relationships for inactivation, production of DNA-double strand-breaks and DNA-protein cross-links are presented. The corresponding F37-values for inactivation and production of DNA lesions are significantly increased only at 80 K (factor of 4 to 5). The data indicate that the low temperatures that prevail in the outer parts of the Solar System or at the nightside of Mars or the Moon are not sufficiently low to crucially inhibit inactivation by dehydration. Our data place further constraints on the panspermia hypothesis.

  1. Novel Secretion Apparatus Maintains Spore Integrity and Developmental Gene Expression in Bacillus subtilis

    Science.gov (United States)

    Meisner, Jeffrey; Serrano, Monica; Henriques, Adriano O.; Moran, Charles P.; Rudner, David Z.

    2009-01-01

    Sporulation in Bacillus subtilis involves two cells that follow separate but coordinately regulated developmental programs. Late in sporulation, the developing spore (the forespore) resides within a mother cell. The regulation of the forespore transcription factor σG that acts at this stage has remained enigmatic. σG activity requires eight mother-cell proteins encoded in the spoIIIA operon and the forespore protein SpoIIQ. Several of the SpoIIIA proteins share similarity with components of specialized secretion systems. One of them resembles a secretion ATPase and we demonstrate that the ATPase motifs are required for σG activity. We further show that the SpoIIIA proteins and SpoIIQ reside in a multimeric complex that spans the two membranes surrounding the forespore. Finally, we have discovered that these proteins are all required to maintain forespore integrity. In their absence, the forespore develops large invaginations and collapses. Importantly, maintenance of forespore integrity does not require σG. These results support a model in which the SpoIIIA-SpoIIQ proteins form a novel secretion apparatus that allows the mother cell to nurture the forespore, thereby maintaining forespore physiology and σG activity during spore maturation. PMID:19609349

  2. Detection of Agar, by Analysis of Sugar Markers, Associated with Bacillus Anthracis Spores, After Culture

    Energy Technology Data Exchange (ETDEWEB)

    Wunschel, David S.; Colburn, Heather A.; Fox, Alvin; Fox, Karen F.; Harley, William M.; Wahl, Jon H.; Wahl, Karen L.

    2008-08-01

    Detection of small quantities of agar associated with spores of Bacillus anthracis could provide key information regarding its source or growth characteristics. Agar, widely used in growth of bacteria on solid surfaces, consists primarily of repeating polysaccharide units of 3,6-anhydro-L-galactose (AGal) and galactose (Gal) with sulfated and O-methylated galactoses present as minor constituents. Two variants of the alditol acetate procedure were evaluated for detection of potential agar markers associated with spores. The first method employed a reductive hydrolysis step, to stabilize labile anhydrogalactose, by converting to anhydrogalactitol. The second eliminated the reductive hydrolysis step simplifying the procedure. Anhydrogalactitol, derived from agar, was detected using both derivatization methods followed by gas chromatography-mass spectrometry (GC-MS) analysis. However, challenges with artefactual background (reductive hydrolysis) or marker destruction (hydrolysis) lead to the search for alternative sugar markers. A minor agar component, 6-O-methyl galactose (6-O-M gal), was readily detected in agar-grown but not broth-grown bacteria. Detection was optimized by the use of gas chromatography-tandem mass spectrometry (GC-MS-MS). With appropriate choice of sugar marker and analytical procedure, detection of sugar markers for agar has considerable potential in microbial forensics.

  3. Dna stability and survival of bacillus subtilis spores in extreme dryness

    Science.gov (United States)

    Dose, Klaus; Gill, Markus

    1995-06-01

    The inactivation of Bacillus subtilis spores during long-term exposure (up to several months) to extreme dryness (especially vacuum) is strain-dependent, through only to a small degree. During a first phase (lasting about four days) monolayers of spores lose about 20% of their viability, regardless of the strain studied. During this phase loss in viability can be equally attributed both to damages of hydrophobic structures (membranes and proteins) and DNA. During a second phase lasting for the remaining time of experimental observation (weeks, months and years) the loss in viability is slowed. A viability of 55% to 75% (depending on the strain) is attained after a total exposure of 36 days. The loss in viability during the second phase can be correlated with the occurrence of DNA double strand breaks. Also covalent DNA-protein cross-links are formed by vacuum exposure. If the protein moiety of these cross-links is degraded by proteinase K-treatment in vitro additional DNA double strand breaks result. The data are also discussed with respect to survival on Mars and in near Earth orbits.

  4. Forensic application of microbiological culture analysis to identify mail intentionally contaminated with Bacillus anthracis spores.

    Science.gov (United States)

    Beecher, Douglas J

    2006-08-01

    The discovery of a letter intentionally filled with dried Bacillus anthracis spores in the office of a United States senator prompted the collection and quarantine of all mail in congressional buildings. This mail was subsequently searched for additional intentionally contaminated letters. A microbiological sampling strategy was used to locate heavy contamination within the 642 separate plastic bags containing the mail. Swab sampling identified 20 bags for manual and visual examination. Air sampling within the 20 bags indicated that one bag was orders of magnitude more contaminated than all the others. This bag contained a letter addressed to Senator Patrick Leahy that had been loaded with dried B. anthracis spores. Microbiological sampling of compartmentalized batches of mail proved to be efficient and relatively safe. Efficiency was increased by inoculating culture media in the hot zone rather than transferring swab samples to a laboratory for inoculation. All mail sampling was complete within 4 days with minimal contamination of the sampling environment or personnel. However, physically handling the intentionally contaminated letter proved to be exceptionally hazardous, as did sorting of cross-contaminated mail, which resulted in generation of hazardous aerosol and extensive contamination of protective clothing. Nearly 8 x 10(6) CFU was removed from the most highly cross-contaminated piece of mail found. Tracking data indicated that this and other heavily contaminated envelopes had been processed through the same mail sorting equipment as, and within 1 s of, two intentionally contaminated letters.

  5. Mucosal immunity induced by gliadin-presenting spores of Bacillus subtilis in HLA-DQ8-transgenic mice.

    Science.gov (United States)

    Bonavita, Roberta; Isticato, Rachele; Maurano, Francesco; Ricca, Ezio; Rossi, Mauro

    2015-06-01

    The induction of mucosal immunity requires efficient antigen delivery and adjuvant systems. Probiotic bacterial strains are considered to be very promising tools to address both of these needs. In particular, Bacillus subtilis spores are currently under investigation as a long-lived, protease-resistant adjuvant system for different antigens. Furthermore, a non-recombinant approach has been developed based on the stable adsorption of antigen on the spore surface. In the present study, we explored this strategy as a means of modulating the immune response to wheat gliadin, the triggering agent of celiac disease (CD), an enteropathy driven by inflammatory CD4(+) T cells. Gliadin adsorption was tested on untreated or autoclaved wild-type (wt) and mutant (cotH or cotE) spores. We found that gliadin was stably and maximally adsorbed by autoclaved wt spores. We then tested the immune properties of the spore-adsorbed gliadin in HLA-DQ8-transgenic mice, which express one of the two HLA heterodimers associated with CD. In vitro, spore-adsorbed gliadin was efficiently taken up by mouse dendritic cells (DCs). Interestingly, gliadin-pulsed DCs efficiently stimulated splenic CD4(+) T cells from mice immunised with spore-adsorbed gliadin. Nasal pre-dosing with spore-adsorbed gliadin failed to down-regulate the ongoing cellular response in gliadin-sensitised DQ8 mice. Notably, naïve mice inoculated intranasally with multiple doses of spore-adsorbed gliadin developed an intestinal antigen-specific CD4(+) T cell-mediated response. In conclusion, our data highlight the ability of spore-adsorbed gliadin to elicit a T-cell response in the gut that could be exploitable for developing immune strategies in CD.

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

    Science.gov (United States)

    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.

  7. Mathematical relationships between spore concentrations, delta-endotoxin levels, and entomotoxicity of Bacillus thuringiensis preparations produced in different fermentation media.

    Science.gov (United States)

    Vu, Khanh Dang; Tyagi, R D; Surampalli, R Y; Valéro, J R

    2012-11-01

    Mathematic relationships between spore concentrations, delta-endotoxin concentrations and entomotoxicity (Tx) of Bacillus thuringiensis var. kurstaki HD-1 (Btk HD-1) preparations produced in six different media were analysed. The relationship between delta-endotoxin and spore concentration and SpTx-spore (specific Tx per 1000 spore) and spore concentration produced in the different media (starch industry wastewater (SIW) with total solids (TS) concentration of 15g/L, SIW with TS of 30g/L, SIW supplemented with 0.2% (w/v) colloidal chitin, SIW supplemented with 1.25% (w/v) cornstarch and 0.2% (v/v) Tween 80, secondary sludge, and semi-synthetic medium) strictly followed the Power law. Tx and delta endotoxin concentration followed the exponential relation whereas a definite relation between Tx and spore concentration could not be established. Spore and delta-endotoxin produced at the early time (12h) during fermentation might be more toxic than those produced during latter period of fermentation irrespective of media used. Tx and delta-endotoxin concentration exhibited a semi-log linear relationship. Based on these findings, delta-endotoxin concentration can be determined rapidly to monitor the progress of the biopesticide production process.

  8. Survival and Germinability of Bacillus subtilis Spores Exposed to Simulated Mars Solar Radiation: Implications for Life Detection and Planetary Protection

    Science.gov (United States)

    Tauscher, Courtney; Schuerger, Andrew C.; Nicholson, Wayne L.

    2006-08-01

    Bacterial spores have been considered as microbial life that could survive interplanetary transport by natural impact processes or human spaceflight activity. Deposition of terrestrial microbes or their biosignature molecules onto the surface of Mars could negatively impact life detection experiments and planetary protection measures. Simulated Mars solar radiation, particularly the ultraviolet component, has been shown to reduce spore viability, but its effect on spore germination and resulting production of biosignature molecules has not been explored. We examined the survival and germinability of Bacillus subtilis spores exposed to simulated martian conditions that include solar radiation. Spores of B. subtilis that contain luciferase resulting from expression of an sspB-luxAB gene fusion were deposited on aluminum coupons to simulate deposition on spacecraft surfaces and exposed to simulated Mars atmosphere and solar radiation. The equivalent of 42 min of simulated Mars solar radiation exposure reduced spore viability by nearly 3 logs, while germination-induced bioluminescence, a measure of germination metabolism, was reduced by less than 1 log. The data indicate that spores can retain the potential to initiate germination-associated metabolic processes and produce biological signature molecules after being rendered nonviable by exposure to Mars solar radiation.

  9. Bacillus subtilis spores expressing the VP28 antigen: a potential oral treatment to protect Litopenaeus vannamei against white spot syndrome.

    Science.gov (United States)

    Nguyen, Anh T V; Pham, Cuong K; Pham, Huong T T; Pham, Hang L; Nguyen, Anh H; Dang, Lua T; Huynh, Hong A; Cutting, Simon M; Phan, Tuan-Nghia

    2014-09-01

    The envelope protein VP28 of white spot syndrome virus (WSSV) is considered a candidate antigen for use in a potential vaccine to this important shrimp pathogen (the cause of white spot syndrome, WSS). Here, we used spores of Bacillus subtilis to display VP28 on the spore surface. Trials were conducted to evaluate their ability to protect shrimps against WSSV infection. The gene cotB-vp28 was integrated into the chromosome of the laboratory strain B. subtilis PY79, and expression of CotB-VP28 was detected by Western blotting and immunofluorescence. Expression of CotB-VP28 was equivalent to 1000 molecules per spore. PY79 and CotB-VP28 spores were mixed with pellets for feeding of whiteleg shrimps (Litopenaeus vannamei), followed by WSSV challenge. Superoxidase dismutase (SOD), phenoloxidase activities and mortality rates of the two shrimp groups were evaluated. Groups fed with PY79 and CotB-VP28 spores at day 7 had increased SOD activities of 29% and increased phenoloxidase activities of 15% and 33%, respectively, compared to those of the control group. Fourteen days postchallenge, 35% of vaccinated shrimps had died compared to 49% of those fed naked spores (PY79) and 66% untreated, unchallenged animals. These data suggest that spores expressing VP28 have potential as a prophylactic treatment of WSS.

  10. Evaluation of sampling methods for Bacillus spore-contaminated HVAC filters.

    Science.gov (United States)

    Calfee, M Worth; Rose, Laura J; Tufts, Jenia; Morse, Stephen; Clayton, Matt; Touati, Abderrahmane; Griffin-Gatchalian, Nicole; Slone, Christina; McSweeney, Neal

    2014-01-01

    The objective of this study was to compare an extraction-based sampling method to two vacuum-based sampling methods (vacuum sock and 37mm cassette filter) with regards to their ability to recover Bacillus atrophaeus spores (surrogate for Bacillus anthracis) from pleated heating, ventilation, and air conditioning (HVAC) filters that are typically found in commercial and residential buildings. Electrostatic and mechanical HVAC filters were tested, both without and after loading with dust to 50% of their total holding capacity. The results were analyzed by one-way ANOVA across material types, presence or absence of dust, and sampling device. The extraction method gave higher relative recoveries than the two vacuum methods evaluated (p≤0.001). On average, recoveries obtained by the vacuum methods were about 30% of those achieved by the extraction method. Relative recoveries between the two vacuum methods were not significantly different (p>0.05). Although extraction methods yielded higher recoveries than vacuum methods, either HVAC filter sampling approach may provide a rapid and inexpensive mechanism for understanding the extent of contamination following a wide-area biological release incident.

  11. Ultra high pressure homogenization (UHPH) inactivation of Bacillus amyloliquefaciens spores in phosphate buffered saline (PBS) and milk

    OpenAIRE

    Dong, Peng; Erika S. Georget; Aganovic, Kemal; Heinz, Volker; Mathys, Alexander

    2015-01-01

    Ultra high pressure homogenization (UHPH) opens up new areas for dynamic high pressure assisted thermal sterilization of liquids. Bacillus amyloliquefaciens spores are resistant to high isostatic pressure and temperature and were suggested as potential surrogate for high pressure thermal sterilization validation. B. amyloliquefaciens spores suspended in PBS buffer (0.01 M, pH 7.0), low fat milk (1.5%, pH 6.7), and whole milk (3.5%, pH 6.7) at initial concentration of ~106 CFU/mL were subjecte...

  12. Efficacy of gaseous chlorine dioxide in inactivating Bacillus cereus spores attached to and in a biofilm on stainless steel.

    Science.gov (United States)

    Nam, Hyegyeong; Seo, Hyun-Sun; Bang, Jihyun; Kim, Hoikyung; Beuchat, Larry R; Ryu, Jee-Hoon

    2014-10-01

    We evaluated the lethal activity of gaseous chlorine dioxide (ClO2) against Bacillus cereus spores attached to and in biofilm formed on a stainless steel surface. Aqueous ClO2 was prepared by mixing sulfuric acid (5% w/v) with sodium chlorite (10mg/mL), and gaseous ClO2 was produced by vaporization of aqueous ClO2 in an air-tight container. The concentration of gaseous ClO2 in the air within the container increased rapidly at first but gradually decreased over time. The lethality of gaseous ClO2 against B. cereus spores attached to stainless steel coupons (SSCs) and in biofilm formed by the pathogen on SSCs was determined. The B. cereus spores attached to SSCs (5.3±0.1logCFU/coupon) were completely inactivated within 1h at 25°C when treated with gaseous ClO2 (peak concentration: 115.3±5.0 parts per million [ppm]). The total number of vegetative cells and spores in biofilm formed by B. cereus on SSCs was 5.9±0.3logCFU/coupon; the spore count was 5.3±0.1logCFU/coupon. The vegetative cells and spores in biofilm were completely inactivated within 6h (peak concentration: 115.3±5.0ppm). Results show that B. cereus spores in biofilms are more resistant to gaseous ClO2 than are attached spores not in biofilms. Gaseous ClO2 was, nevertheless, very effective in killing B. cereus spores in biofilm on the surface of stainless steel. Results show promise for application of gaseous ClO2 to enhance the microbiological safety of foods that may come in contact with stainless steel and possibly other hard surfaces on which B. cereus biofilms have formed.

  13. A Cumulative Spore Killing Approach: Synergistic Sporicidal Activity of Dilute Peracetic Acid and Ethanol at Low pH Against Clostridium difficile and Bacillus subtilis Spores

    Science.gov (United States)

    Nerandzic, Michelle M.; Sankar C, Thriveen; Setlow, Peter; Donskey, Curtis J.

    2016-01-01

    Background. Alcohol-based hand sanitizers are the primary method of hand hygiene in healthcare settings, but they lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We previously demonstrated that acidification of ethanol induced rapid sporicidal activity, resulting in ethanol formulations with pH 1.5–2 that were as effective as soap and water washing in reducing levels of C difficile spores on hands. We hypothesized that the addition of dilute peracetic acid (PAA) to acidified ethanol would enhance sporicidal activity while allowing elevation of the pH to a level likely to be well tolerated on skin (ie, >3). Methods. We tested the efficacy of acidified ethanol solutions alone or in combination with PAA against C difficile and Bacillus subtilis spores in vitro and against nontoxigenic C difficile spores on hands of volunteers. Results. Acidification of ethanol induced rapid sporicidal activity against C difficile and to a lesser extent B subtilis. The addition of dilute PAA to acidified ethanol resulted in synergistic enhancement of sporicidal activity in a dose-dependent fashion in vitro. On hands, the addition of 1200–2000 ppm PAA enhanced the effectiveness of acidified ethanol formulations, resulting in formulations with pH >3 that were as effective as soap and water washing. Conclusions. Acidification and the addition of dilute PAA induced rapid sporicidal activity in ethanol. Our findings suggest that it may be feasible to develop effective sporicidal ethanol formulations that are safe and tolerable on skin. PMID:26885539

  14. A Cumulative Spore Killing Approach: Synergistic Sporicidal Activity of Dilute Peracetic Acid and Ethanol at Low pH Against Clostridium difficile and Bacillus subtilis Spores.

    Science.gov (United States)

    Nerandzic, Michelle M; Sankar C, Thriveen; Setlow, Peter; Donskey, Curtis J

    2016-01-01

    Background.  Alcohol-based hand sanitizers are the primary method of hand hygiene in healthcare settings, but they lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We previously demonstrated that acidification of ethanol induced rapid sporicidal activity, resulting in ethanol formulations with pH 1.5-2 that were as effective as soap and water washing in reducing levels of C difficile spores on hands. We hypothesized that the addition of dilute peracetic acid (PAA) to acidified ethanol would enhance sporicidal activity while allowing elevation of the pH to a level likely to be well tolerated on skin (ie, >3). Methods.  We tested the efficacy of acidified ethanol solutions alone or in combination with PAA against C difficile and Bacillus subtilis spores in vitro and against nontoxigenic C difficile spores on hands of volunteers. Results.  Acidification of ethanol induced rapid sporicidal activity against C difficile and to a lesser extent B subtilis. The addition of dilute PAA to acidified ethanol resulted in synergistic enhancement of sporicidal activity in a dose-dependent fashion in vitro. On hands, the addition of 1200-2000 ppm PAA enhanced the effectiveness of acidified ethanol formulations, resulting in formulations with pH >3 that were as effective as soap and water washing. Conclusions.  Acidification and the addition of dilute PAA induced rapid sporicidal activity in ethanol. Our findings suggest that it may be feasible to develop effective sporicidal ethanol formulations that are safe and tolerable on skin.

  15. Applicability of UV resistant Bacillus pumilus spore as a human adenovirus surrogate for evaluating the effectiveness of virus inactivation in low-pressure UV treatment systems

    Data.gov (United States)

    U.S. Environmental Protection Agency — Data set includes UV dose, and Bacillus pumilus spore plate counts in colony forming units. This dataset is associated with the following publication: Boczek , L.,...

  16. 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.

    Science.gov (United States)

    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

  17. Detection of Bacillus anthracis spores by super-paramagnetic lateral-flow immunoassays based on "Road Closure".

    Science.gov (United States)

    Wang, Dian-Bing; Tian, Bo; Zhang, Zhi-Ping; Wang, Xu-Ying; Fleming, Joy; Bi, Li-Jun; Yang, Rui-Fu; Zhang, Xian-En

    2015-05-15

    Detection of Bacillus anthracis in the field, whether as a natural infection or as a biothreat remains challenging. Here we have developed a new lateral-flow immunochromatographic assay (LFIA) for B. anthracis spore detection based on the fact that conjugates of B. anthracis spores and super-paramagnetic particles labeled with antibodies will block the pores of chromatographic strips and form retention lines on the strips, instead of the conventionally reported test lines and control lines in classic LFIA. As a result, this new LFIA can simultaneously realize optical, magnetic and naked-eye detection by analyzing signals from the retention lines. As few as 500-700 pure B. anthracis spores can be recognized with CV values less than 8.31% within 5 min of chromatography and a total time of 20 min. For powdery sample tests, this LFIA can endure interference from 25% (w/v) milk, 10% (w/v) baking soda and 10% (w/v) starch without any sample pre-treatment, and has a corresponding detection limit of 6×10(4) spores/g milk powder, 2×10(5) spores/g starch and 5×10(5) spores/g baking soda. Compared with existing methods, this new approach is very competitive in terms of sensitivity, specificity, cost and ease of operation. This proof-of-concept study can also be extended for detection of many other large-sized analytes.

  18. Quantifying the effect of sorbic acid, heat and combination of both on germination and outgrowth of Bacillus subtilis spores at single cell resolution

    NARCIS (Netherlands)

    R. Pandey; G.H. Pieper; A. ter Beek; N.O.E. Vischer; J.P.P.M. Smelt; E.M.M. Manders; S. Brul

    2015-01-01

    Bacillus subtilis spores are a problem for the food industry as they are able to survive preservation processes. The spores often reside in food products, where their inherent protection against various stress treatments causes food spoilage. Sorbic acid is widely used as a weak acid preservative in

  19. Quantification of the impact of single and multiple mild stresses on outgrowth heterogeneity of Bacillus cereus spores.

    Science.gov (United States)

    van Melis, C C J; den Besten, H M W; Nierop Groot, M N; Abee, T

    2014-05-02

    Outgrowth heterogeneity of bacterial spore populations complicates both prediction and efficient control of spore outgrowth. In this study, the impact of mild preservation stresses on outgrowth of Bacillus cereus ATCC 14579 spores was quantified during the first stages of outgrowth. Heterogeneity in outgrowth of heat-treated (90°C for 10 min) and non-heat-treated germinated single spores to the maximum micro-colony stage of 256 cells was assessed by direct imaging on Anopore strips, placed on BHI plates at pH7 and pH5.5, without and with added NaCl or sorbic acid (HSA). At pH7 non-heated and heat-treated germinated spores required 6h to reach the maximum microcolony stage with limited heterogeneity, and these parameters were only slightly affected with both types of spores when incubated at pH7 with added NaCl. Notably, the most pronounced effects were observed during outgrowth of spores at pH5.5 without and with added NaCl or HSA. Non-heat-treated germinated spores showed again efficient outgrowth with limited heterogeneity reaching the maximum microcolony stage after 6h at pH5.5, which increased to 12h and 16 h with added NaCl and HSA, respectively. In contrast, heat-treated spores displayed a strong delay between initial germination and swelling and further outgrowth at pH5.5, resulting in large heterogeneity and low numbers of fastest growers reaching the maximum microcolony stage after 10, 12 and 24h, without and with added NaCl or HSA, respectively. This work shows that Anopore technology provides quantitative information on the impact of combined preservation stresses on outgrowth of single spores, showing that outgrowth of germinated heat-treated spores is significantly affected at pH5.5 with a large fraction of spores arrested in the early outgrowth stage, and with outgrowing cells showing large heterogeneity with only a small fraction committed to relatively fast outgrowth.

  20. Investigation of UV-TiO2 photocatalysis and its mechanism in Bacillus subtilis spore inactivation.

    Science.gov (United States)

    Zhang, Yiqing; Zhou, Lingling; Zhang, Yongji

    2014-09-01

    The inactivation levels of Bacillus subtilis spores for various disinfection processes (ultraviolet (UV), TiO2 and UV-TiO2) were compared. The results showed that the inactivation effect of B. subtilis spores by UV treatment alone was far below that for bacteria without endospores. TiO2 alone in the dark, as a control experiment, exhibited almost no inactivation effect. Compared with UV treatment alone, the inactivation effect increased significantly with the addition of TiO2. Increases of the UV irradiance and TiO2 concentration both contributed to the increase of the inactivation effect. Lipid peroxidation was found to be the underlying mechanism of inactivation. Malondialdehyde (MDA), the degradation product of lipid peroxidation, was used as an index to determine the extent of the reaction. The MDA concentration surged surprisingly to 3.24nmol/mg dry cell with the combination disinfection for 600sec (0.10mW/cm(2) irradiance and 10mg/L TiO2). In contrast, for UV alone or TiO2 in the dark, the MDA concentration was 0.38 and 0.25nmol/mg dry cell, respectively, under the same conditions. This indicated that both UV and TiO2 were essential for lipid peroxidation. Changes in cell ultrastructure were observed by transmission electron microscopy. The cell membrane was heavily damaged and cellular contents were completely lysed with the UV-TiO2 process, suggesting that lipid peroxidation was the root of the enhancement in inactivation efficiency.

  1. Inactivation of Bacillus subtilis spores using various combinations of ultraviolet treatment with addition of hydrogen peroxide.

    Science.gov (United States)

    Zhang, Yiqing; Zhou, Lingling; Zhang, Yongji; Tan, Chaoqun

    2014-01-01

    This study aims at comparing the inactivation of Bacillus subtilis spores by various combinations of UV treatment and hydrogen peroxide (H2O2) addition. The combinations included sequential (UV-H2O2, H2O2-UV) and simultaneous (UV/H2O2) processes. Results showed that B. subtilis spores achieved a certain inactivation effect through UV treatment. However, hardly any inactivation effect by H2O2 alone was observed. H2O2 had a significant synergetic effect when combined with UV treatment, while high irradiance and H2O2 concentration both favored the reaction. When treated with 0.60 mm H2O2 and 113.0 μW/cm(2) UV irradiance for 6 min, the simultaneous UV/H2O2 treatment showed significantly improved disinfection effect (4.13 log) compared to that of UV-H2O2 (3.03 log) and H2O2-UV (2.88 log). The relationship between the inactivation effect and the exposure time followed a typical pseudo-first-order kinetics model. The pseudo-first-order rate constants were 0.478, 0.447 and 0.634 min(-1), for the UV-H2O2, H2O2-UV and UV/H2O2 processes, respectively, further confirming the optimal disinfection effect of the UV/H2O2 process. The disinfection could be ascribed to the OH radicals, as verified by the level of para-chlorobenzoic acid (pCBA).

  2. Ultra high pressure homogenization (UHPH) inactivation of Bacillus amyloliquefaciens spores in phosphate buffered saline (PBS) and milk

    Science.gov (United States)

    Dong, Peng; Georget, Erika S.; Aganovic, Kemal; Heinz, Volker; Mathys, Alexander

    2015-01-01

    Ultra high pressure homogenization (UHPH) opens up new areas for dynamic high pressure assisted thermal sterilization of liquids. Bacillus amyloliquefaciens spores are resistant to high isostatic pressure and temperature and were suggested as potential surrogate for high pressure thermal sterilization validation. B. amyloliquefaciens spores suspended in PBS buffer (0.01 M, pH 7.0), low fat milk (1.5%, pH 6.7), and whole milk (3.5%, pH 6.7) at initial concentration of ~106 CFU/mL were subjected to UHPH treatments at 200, 300, and 350 MPa with an inlet temperature at ~80°C. Thermal inactivation kinetics of B. amyloliquefaciens spores in PBS and milk were assessed with thin wall glass capillaries and modeled using first-order and Weibull models. The residence time during UHPH treatments was estimated to determine the contribution of temperature to spore inactivation by UHPH. No sublethal injury was detected after UHPH treatments using sodium chloride as selective component in the nutrient agar medium. The inactivation profiles of spores in PBS buffer and milk were compared and fat provided no clear protective effect for spores against treatments. Treatment at 200 MPa with valve temperatures lower than 125°C caused no reduction of spores. A reduction of 3.5 log10CFU/mL of B. amyloliquefaciens spores was achieved by treatment at 350 MPa with a valve temperature higher than 150°C. The modeled thermal inactivation and observed inactivation during UHPH treatments suggest that temperature could be the main lethal effect driving inactivation. PMID:26236296

  3. Ultra high pressure homogenization (UHPH) inactivation of Bacillus amyloliquefaciens spores in phosphate buffered saline (PBS) and milk.

    Science.gov (United States)

    Dong, Peng; Georget, Erika S; Aganovic, Kemal; Heinz, Volker; Mathys, Alexander

    2015-01-01

    Ultra high pressure homogenization (UHPH) opens up new areas for dynamic high pressure assisted thermal sterilization of liquids. Bacillus amyloliquefaciens spores are resistant to high isostatic pressure and temperature and were suggested as potential surrogate for high pressure thermal sterilization validation. B. amyloliquefaciens spores suspended in PBS buffer (0.01 M, pH 7.0), low fat milk (1.5%, pH 6.7), and whole milk (3.5%, pH 6.7) at initial concentration of ~10(6) CFU/mL were subjected to UHPH treatments at 200, 300, and 350 MPa with an inlet temperature at ~80°C. Thermal inactivation kinetics of B. amyloliquefaciens spores in PBS and milk were assessed with thin wall glass capillaries and modeled using first-order and Weibull models. The residence time during UHPH treatments was estimated to determine the contribution of temperature to spore inactivation by UHPH. No sublethal injury was detected after UHPH treatments using sodium chloride as selective component in the nutrient agar medium. The inactivation profiles of spores in PBS buffer and milk were compared and fat provided no clear protective effect for spores against treatments. Treatment at 200 MPa with valve temperatures lower than 125°C caused no reduction of spores. A reduction of 3.5 log10CFU/mL of B. amyloliquefaciens spores was achieved by treatment at 350 MPa with a valve temperature higher than 150°C. The modeled thermal inactivation and observed inactivation during UHPH treatments suggest that temperature could be the main lethal effect driving inactivation.

  4. Administration of Bacillus coagulans in calves: recovery from faecal samples and evaluation of functional aspects of spores.

    Science.gov (United States)

    Ripamonti, Barbara; Agazzi, Alessandro; Baldi, Antonella; Balzaretti, Claudia; Bersani, Carla; Pirani, Silvia; Rebucci, Raffaella; Savoini, Giovanni; Stella, Simone; Stenico, Alberta; Domeneghini, Cinzia

    2009-12-01

    An investigation was carried out into the recovery from calf faeces of Bacillus coagulans spores added to the feed as probiotic. For this purpose, Bacillus coagulans spores (9 log₁₀ CFU g⁻¹) were given daily to 10 calves during the whole farming periods; another 10 calves acted as controls. Throughout the trial the faecal spore counts were significantly (P < 0.01) higher in the treated group than in the controls (averaging 2.1 x 10⁵ vs 3.7 x 10⁴ CFU g⁻¹). Bacterial cells were recovered from faecal samples and ribotyping matched the strain isolated from faecal sample to the clone administered to the animals. In addition, the recovered cells were found to maintain their functionality aspects of acid production, survival in artificial gastric juice and in the presence of bile, and attachment to human intestinal epithelial cells. The results further elucidate the fate of spore formers administered to calves, and this will help in the development of new species-specific nutritional strategies.

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

    Science.gov (United States)

    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.

  6. Synergistic effect of electrolyzed water and citric Acid against bacillus cereus cells and spores on cereal grains.

    Science.gov (United States)

    Park, Young Bae; Guo, Jin Yong; Rahman, S M E; Ahn, Juhee; Oh, Deog-Hwan

    2009-01-01

    The effects of acidic electrolyzed water (AcEW), alkaline electrolyzed water (AlEW), 100 ppm sodium hypochlorite (NaClO), and 1% citric acid (CA) alone, and combinations of AcEW with 1% CA (AcEW + CA) and AlEW with 1% CA (AlEW + CA) against Bacillus cereus vegetative cells and spores was evaluated as a function of temperature (25, 30, 40, 50, or 60 degrees C) and dipping time (3 or 6 h). A 3-strain cocktail of Bacillus cereus cells or spores of approximately 10(7) CFU/g was inoculated in various cereal grains (brown rice, Job's tear rice, glutinous rice, and barley rice). B. cereus vegetative cells and spores were more rapidly inactivated at 40 degrees C than at 25 degrees C. Regardless of the dipping time, all treatments reduced the numbers of B. cereus vegetative cells and spore by more than 1 log CFU/g, except the deionized water (DIW), which showed approximately 0.7 log reduction. The reductions of B. cereus cells increased with increasing dipping temperature (25 to 60 degrees C). B. cereus vegetative cells were much more sensitive to the combined treatments than spores. The effectiveness of the combined electrolyzed water (EW) and 1% CA was considerable in inhibiting B. cereus on cereal grains. The application of combined EW and CA for controlling B. cereus cells and spores on cereal grains has not been previously reported. Therefore, the synergistic effect of EW and CA may provide a valuable insight on reducing foodborne pathogens on fruits, vegetables, and cereal grains.

  7. Inactivation of Spores of Bacillus Species by Wet Heat: Studies on Single Spores Using Laser Tweezers Taman Spectroscopy

    Science.gov (United States)

    2013-02-01

    germination using phase contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers, Nature Protocols , (04 2011): . doi: 05/11...multiple individual spores [ Nature Protocols , 6, 625 (2011)]. (1e) We developed a multiple-trap laser tweezers Raman spectroscopy (LTRS) array for

  8. The prevalence and control of Bacillus and related spore-forming bacteria in the dairy industry

    Directory of Open Access Journals (Sweden)

    Nidhi eGopal

    2015-12-01

    Full Text Available Milk produced in udder cells is sterile but due to its high nutrient content, it can be a good growth substrate for contaminating bacteria. The quality of milk is monitored via somatic cell counts and total bacterial counts, with prescribed regulatory limits to ensure quality and safety. Bacterial contaminants can cause disease, or spoilage of milk and its secondary products. Aerobic spore-forming bacteria, such as those from the genera Sporosarcina, Paenisporosarcina, Brevibacillus, Paenibacillus, Geobacillus and Bacillus, are a particular concern in this regard as they are able to survive industrial pasteurisation and form biofilms within pipes and stainless steel equipment. These single or multiple-species biofilms become a reservoir of spoilage microorganisms and a cycle of contamination can be initiated. Indeed, previous studies have highlighted that these microorganisms are highly prevalent in dead ends, corners, cracks, crevices, gaskets, valves and the joints of stainless steel equipment used in the dairy manufacturing plants. Hence, adequate monitoring and control measures are essential to prevent spoilage and ensure consumer safety. Common controlling approaches include specific cleaning-in-place processes, chemical and biological biocides and other novel methods. In this review, we highlight the problems caused by these microorganisms, and discuss issues relating to their prevalence, monitoring thereof and control with respect to the dairy industry.

  9. Nanosensors having dipicolinic acid imprinted nanoshell for Bacillus cereus spores detection

    Energy Technology Data Exchange (ETDEWEB)

    Gueltekin, Aytac [Trakya University, Department of Chemistry (Turkey); Ersoez, Arzu [Anadolu University, Department of Chemistry, Faculty of Science, Yunusemre Campus (Turkey); Sarioezlue, Nalan Yilmaz [Anadolu University, Department of Biology (Turkey); Denizli, Adil [Hacettepe University, Department of Chemistry (Turkey); Say, Ridvan, E-mail: rsay@anadolu.edu.t [Anadolu University, Department of Chemistry, Faculty of Science, Yunusemre Campus (Turkey)

    2010-08-15

    Molecular imprinted polymers (MIPs) as a recognition element for sensors are increasingly of interest and MIP nanoclusters have started to appear in the literature. In this study, we have proposed a novel thiol ligand-capping method with polymerizable methacryloylamido-cysteine (MAC) attached to gold-silver nanoclusters, reminiscent of a self-assembled monolayer and have reconstructed surface shell by synthetic host polymers based on molecular imprinting method for recognition. In this method, methacryloylamidoantipyrine-terbium ((MAAP){sub 2}-Tb(III)) has been used as a new metal-chelating monomer via metal coordination-chelation interactions and dipicolinic acid (DPA) which is main participant of Bacillus cereus spores used as a model. Nanoshell sensors with templates give a cavity that is selective for DPA. The DPA can simultaneously chelate to Tb(III) metal ion and fit into the shape-selective cavity. Thus, the interaction between Tb(III) ion and free coordination spheres has an effect on the binding ability of the gold-silver nanoclusters nanosensor. The binding affinity of the DPA imprinted nanoclusters has been investigated by using the Langmuir and Scatchard methods, and the respective affinity constants (K{sub affinity}) determined were found to be 1.43 x 10{sup 4} and 9.1 x 10{sup 6} mol L{sup -1}.

  10. Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore.

    Science.gov (United States)

    Tang, Jilin; Krajcikova, Daniela; Zhu, Rong; Ebner, Andreas; Cutting, Simon; Gruber, Hermann J; Barak, Imrich; Hinterdorfer, Peter

    2007-01-01

    Coat assembly in Bacillus subtilis serves as a tractable model for the study of the self-assembly process of biological structures and has a significant potential for use in nano-biotechnological applications. In the present study, the morphology of B. subtilis spores was investigated by magnetically driven dynamic force microscopy (MAC mode atomic force microscopy) under physiological conditions. B. subtilis spores appeared as prolate structures, with a length of 0.6-3 microm and a width of about 0.5-2 microm. The spore surface was mainly covered with bump-like structures with diameters ranging from 8 to 70 nm. Besides topographical explorations, single molecule recognition force spectroscopy (SMRFS) was used to characterize the spore coat protein CotA. This protein was specifically recognized by a polyclonal antibody directed against CotA (anti-CotA), the antibody being covalently tethered to the AFM tip via a polyethylene glycol linker. The unbinding force between CotA and anti-CotA was determined as 55 +/- 2 pN. From the high-binding probability of more than 20% in force-distance cycles it is concluded that CotA locates in the outer surface of B. subtilis spores.

  11. Inhibitory effects of nisin-coated multi-walled carbon nanotube sheet on biofilm formation from Bacillus anthracis spores

    Institute of Scientific and Technical Information of China (English)

    Xiuli Dong; Eric McCoy; Mei Zhang; Liju Yang

    2014-01-01

    Multi-walled carbon nanotube (MWCNT) sheet was fabricated from a drawable MWCNT forest and then deposited on poly(methyl methacrylate) film.The film was further coated with a natural antimicrobial peptide nisin.We studied the effects of nisin coating on the attachment of Bacillus anthracis spores,the germination of attached spores,and the subsequent biofilm formation from attached spores.It was found that the strong adsorptivity and the super hydrophobicity of MWCNTs provided an ideal platform for nisin coating.Nisin coating on MWCNT sheets decreased surface hydrophobicity,reduced spore attachment,and reduced the germination of attached spores by 3.5 fold,and further inhibited the subsequent biofilm formation by 94.6% compared to that on uncoated MWCNT sheet.Nisin also changed the morphology of vegetative cells in the formed biofilm.The results of this study demonstrated that the anti-adhesion and antimicrobial effect of nisin in combination with the physical properties of carbon nanotubes had the potential in producing effective anti-biofilm formation surfaces.

  12. Inhibitory effects of nisin-coated multi-walled carbon nanotube sheet on biofilm formation from Bacillus anthracis spores.

    Science.gov (United States)

    Dong, Xiuli; McCoy, Eric; Zhang, Mei; Yang, Liju

    2014-12-01

    Multi-walled carbon nanotube (MWCNT) sheet was fabricated from a drawable MWCNT forest and then deposited on poly(methyl methacrylate) film. The film was further coated with a natural antimicrobial peptide nisin. We studied the effects of nisin coating on the attachment of Bacillus anthracis spores, the germination of attached spores, and the subsequent biofilm formation from attached spores. It was found that the strong adsorptivity and the super hydrophobicity of MWCNTs provided an ideal platform for nisin coating. Nisin coating on MWCNT sheets decreased surface hydrophobicity, reduced spore attachment, and reduced the germination of attached spores by 3.5 fold, and further inhibited the subsequent biofilm formation by 94.6% compared to that on uncoated MWCNT sheet. Nisin also changed the morphology of vegetative cells in the formed biofilm. The results of this study demonstrated that the anti-adhesion and antimicrobial effect of nisin in combination with the physical properties of carbon nanotubes had the potential in producing effective anti-biofilm formation surfaces.

  13. Identification of Differentially Expressed Genes during Bacillus subtilis Spore Outgrowth in High-Salinity Environments Using RNA Sequencing

    Science.gov (United States)

    Nagler, Katja; Krawczyk, Antonina O.; De Jong, Anne; Madela, Kazimierz; Hoffmann, Tamara; Laue, Michael; Kuipers, Oscar P.; Bremer, Erhard; Moeller, Ralf

    2016-01-01

    In its natural habitat, the soil bacterium Bacillus subtilis often has to cope with fluctuating osmolality and nutrient availability. Upon nutrient depletion it can form dormant spores, which can revive to form vegetative cells when nutrients become available again. While the effects of salt stress on spore germination have been analyzed previously, detailed knowledge on the salt stress response during the subsequent outgrowth phase is lacking. In this study, we investigated the changes in gene expression during B. subtilis outgrowth in the presence of 1.2 M NaCl using RNA sequencing. In total, 402 different genes were upregulated and 632 genes were downregulated during 90 min of outgrowth in the presence of salt. The salt stress response of outgrowing spores largely resembled the osmospecific response of vegetative cells exposed to sustained high salinity and included strong upregulation of genes involved in osmoprotectant uptake and compatible solute synthesis. The σB-dependent general stress response typically triggered by salt shocks was not induced, whereas the σW regulon appears to play an important role for osmoadaptation of outgrowing spores. Furthermore, high salinity induced many changes in the membrane protein and transporter transcriptome. Overall, salt stress seemed to slow down the complex molecular reorganization processes (“ripening”) of outgrowing spores by exerting detrimental effects on vegetative functions such as amino acid metabolism. PMID:27766092

  14. A genetic algorithm-Bayesian network approach for the analysis of metabolomics and spectroscopic data: application to the rapid identification of Bacillus spores and classification of Bacillus species

    Directory of Open Access Journals (Sweden)

    Goodacre Royston

    2011-01-01

    Full Text Available Abstract Background The rapid identification of Bacillus spores and bacterial identification are paramount because of their implications in food poisoning, pathogenesis and their use as potential biowarfare agents. Many automated analytical techniques such as Curie-point pyrolysis mass spectrometry (Py-MS have been used to identify bacterial spores giving use to large amounts of analytical data. This high number of features makes interpretation of the data extremely difficult We analysed Py-MS data from 36 different strains of aerobic endospore-forming bacteria encompassing seven different species. These bacteria were grown axenically on nutrient agar and vegetative biomass and spores were analyzed by Curie-point Py-MS. Results We develop a novel genetic algorithm-Bayesian network algorithm that accurately identifies sand selects a small subset of key relevant mass spectra (biomarkers to be further analysed. Once identified, this subset of relevant biomarkers was then used to identify Bacillus spores successfully and to identify Bacillus species via a Bayesian network model specifically built for this reduced set of features. Conclusions This final compact Bayesian network classification model is parsimonious, computationally fast to run and its graphical visualization allows easy interpretation of the probabilistic relationships among selected biomarkers. In addition, we compare the features selected by the genetic algorithm-Bayesian network approach with the features selected by partial least squares-discriminant analysis (PLS-DA. The classification accuracy results show that the set of features selected by the GA-BN is far superior to PLS-DA.

  15. Infection of Tribolium castaneum with Bacillus thuringiensis: Quantification of Bacterial Replication within Cadavers, Transmission via Cannibalism, and Inhibition of Spore Germination

    Science.gov (United States)

    Milutinović, Barbara; Höfling, Christina; Futo, Momir; Scharsack, Jörn P.

    2015-01-01

    Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen. PMID:26386058

  16. Infection of Tribolium castaneum with Bacillus thuringiensis: quantification of bacterial replication within cadavers, transmission via cannibalism, and inhibition of spore germination.

    Science.gov (United States)

    Milutinović, Barbara; Höfling, Christina; Futo, Momir; Scharsack, Jörn P; Kurtz, Joachim

    2015-12-01

    Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen.

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

    Science.gov (United States)

    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.

  18. SporeWeb : an interactive journey through the complete sporulation cycle of Bacillus subtilis

    NARCIS (Netherlands)

    Eijlander, Robyn T.; Jong, Anne de; Krawczyk, Antonina O.; Holsappel, Siger; Kuipers, Oscar P.

    2014-01-01

    Bacterial spores are a continuous problem for both food-based and health-related industries. Decades of scientific research dedicated towards understanding molecular and gene regulatory aspects of sporulation, spore germination and spore properties have resulted in a wealth of data and information.

  19. Impact of sorbic acid on germination and outgrowth heterogeneity of Bacillus cereus ATCC 14579 spores

    NARCIS (Netherlands)

    Besten, den H.M.W.; Melis, van C.C.J.; Sanders, J.W.; Nierop Groot, M.N.; Abee, T.

    2012-01-01

    Population heterogeneity complicates the predictability of the outgrowth kinetics of individual spores. Flow cytometry sorting and monitoring of the germination and outgrowth of single dormant spores allowed the quantification of acid-induced spore population heterogeneity at pH 5.5 and in the prese

  20. Influence of food matrix on outgrowth heterogeneity of heat damaged Bacillus cereus spores

    NARCIS (Netherlands)

    Warda, A.K.; Besten, den H.M.W.; Sha, N.; Abee, T.; Nierop Groot, M.N.

    2015-01-01

    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

  1. Gamma radiation effect on Bacillus cereus spores inoculated in black pepper; Efeitos da radiacao gama sobre esporos de Bacillus cereus inoculados em pimenta-do-reino

    Energy Technology Data Exchange (ETDEWEB)

    Froehlich, Angela; Axeredo, Raquel M.C.; Vanetti, Maria Cristina D. [Vicosa Univ., MG (Brazil). Dept. de Microbiologia; Villavicencio, Anna Lucia C. H. [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil)]. E-mail: villavic@net.ipen.br

    2000-07-01

    It had been analyzed 37 samples of worn out black pepper and in 85% of these samples was observed the presence of Bacillus cereus in numbers of up to 4,6 x 10{sup 4} UFC/g. The population of aerobic mesofilis bacteria varied of 2,8 x 10{sup 5} the 1,9 x 10{sup 8} UFC/g. The black pepper used during the experiment was evaluated, evidencing the aerobic presence of one aerobic mesofilis microbiota of, approximately, 2,6 x 10{sup 6} UFC/g, consisting, mainly, for species of the Bacillus sort. It was observed that the absence of B. cereus, coliforms, filamentous fungus and leavenings. The evaluation of the irradiation of the black pepper inoculated with 10{sup 6} UFC/g of B. cereus spores of with doses of gamma radiation varying between 2 and 10 kGy evidenced that doses up to 5 kGy had been enough to reduce the counting of, approximately, 10{sup 6} UFC/g of aerobic mesofilis organisms and 10{sup 4} UFC/g of B. cereus spores the not detectable numbers by the used methodology. The dose of reduction decimal (D{sub 10}) for the inoculated B. cereus spores in black pepper was of 1,78 kGy.

  2. Transcriptome analysis of Bacillus thuringiensis spore life, germination and cell outgrowth in a vegetable-based food model.

    Science.gov (United States)

    Bassi, Daniela; Colla, Francesca; Gazzola, Simona; Puglisi, Edoardo; Delledonne, Massimo; Cocconcelli, Pier Sandro

    2016-05-01

    Toxigenic species belonging to Bacillus cereus sensu lato, including Bacillus thuringiensis, cause foodborne outbreaks thanks to their capacity to survive as spores and to grow in food matrixes. The goal of this work was to assess by means of a genome-wide transcriptional assay, in the food isolate B. thuringiensis UC10070, the gene expression behind the process of spore germination and consequent outgrowth in a vegetable-based food model. Scanning electron microscopy and Energy Dispersive X-ray microanalysis were applied to select the key steps of B. thuringiensis UC10070 cell cycle to be analyzed with DNA-microarrays. At only 40 min from heat activation, germination started rapidly and in less than two hours spores transformed in active growing cells. A total of 1646 genes were found to be differentially expressed and modulated during the entire B. cereus life cycle in the food model, with most of the significant genes belonging to transport, transcriptional regulation and protein synthesis, cell wall and motility and DNA repair groups. Gene expression studies revealed that toxin-coding genes nheC, cytK and hblC were found to be expressed in vegetative cells growing in the food model.

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

    Science.gov (United States)

    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.

  4. Bacterial spores as possible contaminants of biomedical materials and devices. [Bacillus anthracis, clostridium botulinum, C. perfringens, C. tetani

    Energy Technology Data Exchange (ETDEWEB)

    Grecz, N.; Kang, T.

    1973-01-01

    Destruction of spores on biomedical devices in drugs, and biologicals is essential for prevention of infection of patients with pathogenic sporeformers. Of particular concern are Clostridium tetani, C. perfringens, C. botulinum, Bacillus anthracis and other sporeforming pathogens. Spores are ubiquitous in nature and contamination of biomedical devices varies depending on manufacturing process, handling, raw materials and other variables. In the last 20 years the number of cases per year of specific notifiable diseases in the United States was as follows: tetanus, 120 to 500 cases, botulism, 7 to 47 cases, and anthrax, 2 to 10 cases. Gas gangrene is caused by a mixed flora consisting predominantly of sporeformers. C botulinum, which usually acts as saprophytic agent of food poisoning, may also initiate pathogenic processes; there are nine cases on record in the United States of botulism wound infections almost half of which ended in death. The spores of these organisms are distinguished by high radiation resistance and their erradication often requires severe radiation treatments. Representative bacterial spores in various suspending media show D/sub 10/ values (dose necessary to destroy 90 percent of a given population) ranging from approximately 0.1 to 0.4 Mrad. Some viruses show D/sub 10/ values up to greater than 1 Mrad. The D/sub 10/-values of spores vary depending on physical, chemical and biological factors. This variability is important in evaluation and selection of biological indicator organisms. Radiation sterilization of biomedical devices and biomedical materials must provide safety from infectious microorganisms including radiation resistant spores and viruses.

  5. Gold nanoparticles having dipicolinic acid imprinted nanoshell for Bacillus cereus spores recognition

    Energy Technology Data Exchange (ETDEWEB)

    Gueltekin, Aytac [Department of Chemistry, Trakya University, Edirne (Turkey); Ersoez, Arzu; Huer, Deniz [Department of Chemistry, Anadolu University, Eskisehir (Turkey); Sarioezlue, Nalan Yilmaz [Department of Biology, Anadolu University, Eskisehir (Turkey); Denizli, Adil [Department of Chemistry, Hacettepe University, Ankara (Turkey); Say, Ridvan, E-mail: rsay@anadolu.edu.tr [Department of Chemistry, Anadolu University, Eskisehir (Turkey); BIBAM (Plant, Drug and Scientific Research Center) Anadolu University (Turkey)

    2009-10-15

    Taking into account the recognition element for sensors linked to molecular imprinted polymers (MIPs), a proliferation of interest has been witnessed by those who are interested in this subject. Indeed, MIP nanoparticles are theme which recently has come to light in the literature. In this study, we have proposed a novel thiol ligand-capping method with polymerizable methacryloylamidocysteine (MAC) attached to gold nanoparticles, reminiscent of a self-assembled monolayer. Furthermore, a surface shell by synthetic host polymers based on molecular imprinting method for recognition has been reconstructed. In this method, methacryloyl iminodiacetic acid-chrome (MAIDA-Cr(III)) has been used as a new metal-chelating monomer via metal coordination-chelation interactions and dipicolinic acid (DPA) which is the main participant of Bacillus cereus spores has been used as a template. Nanoshell sensors with templates produce a cavity that is selective for DPA. The DPA can simultaneously chelate to Cr(III) metal ion and fit into the shape-selective cavity. Thus, the interaction between Cr(III) ion and free coordination spheres has an effect on the binding ability of the gold nanoparticles nanosensor. The interactions between DPA and MIP particles were studied observing fluorescence measurements. DPA addition caused significant decreases in fluorescence intensity because they induced photoluminescence emission from Au nanoparticles through the specific binding to the recognition sites of the crosslinked nanoshell polymer matrix. The binding affinity of the DPA imprinted nanoparticles has been explored by using the Langmuir and Scatchard methods and the analysis of the quenching results has been performed in terms of the Stern-Volmer equation.

  6. Food Sensing: Aptamer-Based Trapping of Bacillus cereus Spores with Specific Detection via Real Time PCR in Milk.

    Science.gov (United States)

    Fischer, Christin; Hünniger, Tim; Jarck, Jan-Hinnerk; Frohnmeyer, Esther; Kallinich, Constanze; Haase, Ilka; Hahn, Ulrich; Fischer, Markus

    2015-09-16

    Aerobic spores pose serious problems for both food product manufacturers and consumers. Milk is particularly at risk and thus an important issue of preventive consumer protection and quality assurance. The spore-former Bacillus cereus is a food poisoning Gram-positive pathogen which mainly produces two different types of toxins, the diarrhea inducing and the emetic toxins. Reliable and rapid analytical assays for the detection of B. cereus spores are required, which could be achieved by combining in vitro generated aptamers with highly specific molecular biological techniques. For the development of routine bioanalytical approaches, already existing aptamers with high affinity to B. cereus spores have been characterized by surface plasmon resonance (SPR) spectroscopy and fluorescence microscopy in terms of their dissociation constants and selectivity. Dissociation constants in the low nanomolar range (from 5.2 to 52.4 nM) were determined. Subsequently, the characterized aptamers were utilized for the establishment and validation of an aptamer-based trapping technique in both milk simulating buffer and milk with fat contents between 0.3 and 3.5%. Thereby, enrichment factors of up to 6-fold could be achieved. It could be observed that trapping protocol and characterized aptamers were fully adaptable to the application in milk. Due to the fact that aptamer selectivity is limited, a highly specific real time PCR assay was utilized following trapping to gain a higher degree of selectivity.

  7. Study of the antibacterial effects of chitosans on Bacillus cereus (and its spores) by atomic force microscopy imaging and nanoindentation

    Energy Technology Data Exchange (ETDEWEB)

    Fernandes, Joao C. [Escola Superior de Biotecnologia, Universidade Catolica Portuguesa, Rua Dr. Antonio Bernardino de Almeida, 4200-072 Porto (Portugal); Eaton, Peter, E-mail: peter.eaton@fc.up.pt [REQUIMTE, Departamento de Quimica, Faculdade de Ciencias da Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto (Portugal); Gomes, Ana M.; Pintado, Manuela E.; Xavier Malcata, F. [Escola Superior de Biotecnologia, Universidade Catolica Portuguesa, Rua Dr. Antonio Bernardino de Almeida, 4200-072 Porto (Portugal)

    2009-07-15

    Bacillus cereus is a Gram-positive, spore-forming bacterium that is widely distributed in nature. Its intrinsic thermal resistance coupled with the extraordinary resistance against common food preservation techniques makes it one of the most frequent food-poisoning microorganisms causing both intoxications and infections. In order to control B. cereus growth/sporulation, and hence minimize the aforementioned hazards, several antimicrobial compounds have been tested. The aim of this work was to assess by atomic force microscopy (AFM) the relationship between the molecular weight (MW) of chitosan and its antimicrobial activity upon both vegetative and resistance forms of B. cereus. The use of AFM imaging studies helped us to understand how chitosans with different MW act differently upon B. cereus. Higher MW chitosans (628 and 100 kDa) surrounded both forms of B. cereus cells by forming a polymer layer-which eventually led to the death of the vegetative form by preventing the uptake of nutrients yet did not affect the spores since these can survive for extended periods without nutrients. Chitooligosaccharides (COS) (<3 kDa), on the other hand, provoked more visible damages in the B. cereus vegetative form-most probably due to the penetration of the cells by the COS. The use of COS by itself on B. cereus spores was not enough for the destruction of a large number of cells, but it may well weaken the spore structure and its ability to contaminate, by inducing exosporium loss.

  8. SirA enforces diploidy by inhibiting the replication initiator DnaA during spore formation in Bacillus subtilis.

    Science.gov (United States)

    Wagner, Jennifer K; Marquis, Kathleen A; Rudner, David Z

    2009-09-01

    How cells maintain their ploidy is relevant to cellular development and disease. Here, we investigate the mechanism by which the bacterium Bacillus subtilis enforces diploidy as it differentiates into a dormant spore. We demonstrate that a sporulation-induced protein SirA (originally annotated YneE) blocks new rounds of replication by targeting the highly conserved replication initiation factor DnaA. We show that SirA interacts with DnaA and displaces it from the replication origin. As a result, expression of SirA during growth rapidly blocks replication and causes cell death in a DnaA-dependent manner. Finally, cells lacking SirA over-replicate during sporulation. These results support a model in which induction of SirA enforces diploidy by inhibiting replication initiation as B. subtilis cells develop into spores.

  9. Morphology and physico-chemical properties of Bacillus spores surrounded or not with an exosporium: consequences on their ability to adhere to stainless steel.

    Science.gov (United States)

    Faille, Christine; Lequette, Yannick; Ronse, Annette; Slomianny, Christian; Garénaux, Estelle; Guerardel, Yann

    2010-10-15

    This study was designed to elucidate the influence of spore properties such as the presence of an exosporium, on their ability to adhere to materials. This analysis was performed on 17 strains belonging to the B. cereus group and to less related Bacillus species. We first demonstrated that spores of the B. cereus group, surrounded by an exosporium, differed in their morphological features such as exosporium size, number of appendages or hair-like nap length. We also found that the saccharidic composition of exosporium differed among strains, e.g. concerning a newly identified rhamnose derivative: the 2,4-O-dimethyl-rhamnose. Conversely, spores of distant Bacillus species shared morphological and physico-chemical properties with B. cereus spores. Some external features were also observed on these spores, such as a thin loose-fitting layer, whose nature is still to be determined, or a thick saccharidic layer (mainly composed of rhamnose and quinovose). The ability of spores to adhere to stainless steel varied among strains, those belonging to the B. cereus group generally being the most adherent. However, the presence of an exosporium is not sufficient to explain the ability of spores to adhere to inanimate surfaces. Indeed, when the 17 strains were compared, hydrophobicity and the number of appendages were the only significant adhesion parameters. Furthermore, the differences in spore adhesion observed within the B. cereus group were related to differences in the number of appendages, the exosporium length and to a lesser extent, the zeta potential.

  10. Enhancement of intrinsic antitumor activity in spore-endotoxin mixtures of Bacillus thuringiensis by exposure to ultraviolet radiation

    Energy Technology Data Exchange (ETDEWEB)

    Zamola, B.; Karminski-Zamola, G.; Fuks, Z.; Kubovic, M. (Zagreb Univ. (Yugoslavia)); Wrishcer, M. (Institut Rudjer Boskovic, Zagreb (Yugoslavia))

    1985-03-01

    Irradiation of spore-endotoxin mixtures from Bacillus thuringiensis cultures at 254 nm (60 ..mu..W cm/sup -2/) enhances their intrinsic antitumor potency as well as that of either component. The extent of enhancement depends on the length of exposure (optimum: 35 min) and may thus be due to photochemical changes of the endotoxin protein or/and to photoproduction of additional compounds with antitumor activity. Antitumor effects, expressed as survival rates of C57BL/6 mice inoculated with Lewis' mouse lung carcinoma and subjected to treatments 24 h later, depended on the number of doses of preparations administered (mixture, separated components).

  11. SirA enforces diploidy by inhibiting the replication initiator DnaA during spore formation in Bacillus subtilis

    OpenAIRE

    Jennifer K. Wagner; Marquis, Kathleen A.; Rudner, David Z.

    2009-01-01

    How cells maintain their ploidy is relevant to cellular development and disease. Here, we investigate the mechanism by which the bacterium Bacillus subtilis enforces diploidy as it differentiates into a dormant spore. We demonstrate that a sporulation-induced protein SirA (originally annotated YneE) blocks new rounds of replication by targeting the highly conserved replication initiation factor DnaA. We show that SirA interacts with DnaA and displaces it from the replication origin. As a resu...

  12. Unlocking the Sporicidal Potential of Ethanol: Induced Sporicidal Activity of Ethanol against Clostridium difficile and Bacillus Spores under Altered Physical and Chemical Conditions

    Science.gov (United States)

    Nerandzic, Michelle M.; Sunkesula, Venkata C. K.; C., Thriveen Sankar; Setlow, Peter; Donskey, Curtis J.

    2015-01-01

    Background Due to their efficacy and convenience, alcohol-based hand sanitizers have been widely adopted as the primary method of hand hygiene in healthcare settings. However, alcohols lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We hypothesized that sporicidal activity could be induced in alcohols through alteration of physical or chemical conditions that have been shown to degrade or allow penetration of spore coats. Principal Findings Acidification, alkalinization, and heating of ethanol induced rapid sporicidal activity against C. difficile, and to a lesser extent Bacillus thuringiensis and Bacillus subtilis. The sporicidal activity of acidified ethanol was enhanced by increasing ionic strength and mild elevations in temperature. On skin, sporicidal ethanol formulations were as effective as soap and water hand washing in reducing levels of C. difficile spores. Conclusions These findings demonstrate that novel ethanol-based sporicidal hand hygiene formulations can be developed through alteration of physical and chemical conditions. PMID:26177038

  13. 40 CFR 180.1011 - Viable spores of the microorganism Bacillus thuringiensis Berliner; exemption from the...

    Science.gov (United States)

    2010-07-01

    ... Bacillus thuringiensis Berliner; exemption from the requirement of a tolerance. 180.1011 Section 180.1011... microorganism Bacillus thuringiensis Berliner; exemption from the requirement of a tolerance. (a) For the... authentic strain of Bacillus thuringiensis Berliner conforming to the morphological and...

  14. Dynamics of Bacillus thuringiensis var. israelensis and Lysinibacillus sphaericus spores in urban catch basins after simultaneous application against mosquito larvae.

    Directory of Open Access Journals (Sweden)

    Valeria Guidi

    Full Text Available Bacillus thuringiensis var. israelensis (Bti and Lysinibacillus sphaericus (Lsph are extensively used in mosquito control programs. These biocides are the active ingredients of a commercial larvicide. Quantitative data on the fate of both Bti and Lsph applied together for the control of mosquitoes in urban drainage structures such as catch basins are lacking. We evaluated the dynamics and persistence of Bti and Lsph spores released through their concomitant application in urban catch basins in southern Switzerland. Detection and quantification of spores over time in water and sludge samples from catch basins were carried out using quantitative real-time PCR targeting both cry4A and cry4B toxin genes for Bti and the binA gene for Lsph. After treatment, Bti and Lsph spores attained concentrations of 3.76 (± 0.08 and 4.13 (± 0.09 log ml(-1 in water, then decreased progressively over time, reaching baseline values. For both Bti and Lsph, spore levels in the order of 10(5 g(-1 were observed in the bottom sludge two days after the treatment and remained constant for the whole test period (275 days. Indigenous Lsph strains were isolated from previously untreated catch basins. A selection of those was genotyped using pulsed field gel electrophoresis of SmaI-digested chromosomal DNA, revealing that a subset of isolates were members of the clonal population of strain 2362. No safety issues related to the use of this biopesticide in the environment have been observed during this study, because no significant increase in the number of spores was seen during the long observation period. The isolation of native Lysinibacillus sphaericus strains belonging to the same clonal population as strain 2362 from catch basins never treated with Lsph-based products indicates that the use of a combination of Bti and Lsph for the control of mosquitoes does not introduce non-indigenous microorganisms in this area.

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

    OpenAIRE

    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 compar...

  16. Etude de la résistance à la chaleur des spores de Bacillus subtilis déshydratées

    OpenAIRE

    Hauck Tiburski, Julia

    2013-01-01

    In response to starvation, species from the genre Bacillus are able to form metabolicallydormant spores which are very resistant to multiple forms of stress. They are found in quitehigh concentrations in some dried foods which, upon rehydration, may lead to food deterioration or food-borne diseases. Moreover, their destruction is rather difficult and mostof the techniques commonly used to treat dry foods result in a very low spore inactivation.The aim of this work is to better understand the ...

  17. Functional and Immunological Analyses of Superoxide Dismutases and Other Spore-Associated Proteins of Bacillus anthracis

    Science.gov (United States)

    2008-08-20

    performed experiments on aerial dispersal of spores in the Guinard Islands near Scotland , a study that ultimately led to observations on the capacity...respiratory route. J. Pathol. Bacteriol. 73:485-494. 188. ROTH , N. G. and D. H. LIVELY. 1956. Germination of spores of certain aerobic bacilli under

  18. Evaluating the transport of bacillus subtilis spores as a potential surrogate for Cryptosporidium parvum Oocysts

    Science.gov (United States)

    The USEPA 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 r...

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

    NARCIS (Netherlands)

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

    2007-01-01

    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

  20. Bacillus cereus and Bacillus thuringiensis spores in Korean rice: prevalence and toxin production as affected by production area and degree of milling.

    Science.gov (United States)

    Kim, Booyoung; Bang, Jihyun; Kim, Hoikyung; Kim, Yoonsook; Kim, Byeong-Sam; Beuchat, Larry R; Ryu, Jee-Hoon

    2014-09-01

    We determined the prevalence of and toxin production by Bacillus cereus and Bacillus thuringiensis in Korean rice as affected by production area and degree of milling. Rough rice was collected from 64 farms in 22 agricultural areas and polished to produce brown and white rice. In total, rice samples were broadly contaminated with B. cereus spores, with no effect of production area. The prevalence and counts of B. cereus spores declined as milling progressed. Frequencies of hemolysin BL (HBL) production by isolates were significantly (P ≤ 0.01) reduced as milling progressed. This pattern corresponded with the presence of genes encoding the diarrheal enterotoxins. The frequency of B. cereus isolates positive for hblC, hblD, or nheB genes decreased as milling progressed. Because most B. cereus isolates from rice samples contained six enterotoxin genes, we concluded that B. cereus in rice produced in Korea is predominantly of the diarrheagenic type. The prevalence of B. thuringiensis in rice was significantly lower than that of B. cereus and not correlated with production area. All B. thuringiensis isolates were of the diarrheagenic type. This study provides information useful for predicting safety risks associated with B. cereus and B. thuringiensis in rough and processed Korean rice.

  1. Crystal structure of the PepSY-containing domain of the YpeB protein involved in germination of Bacillus spores

    OpenAIRE

    2015-01-01

    This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/prot.24868 The crystal structure of the C-terminal domain of the Bacillus megaterium YpeB protein has been solved by X-ray crystallography to 1.80 Å resolution. The full-length protein is essential in stabilising the SleB cortex lytic enzyme in Bacillus spores, and may have a role in regulating SleB activity during spore germination. The YpeB-C crystal structure comprises three t...

  2. Crystal structure of the PepSY-containing domain of the YpeB protein involved in germination of Bacillus spores

    OpenAIRE

    2015-01-01

    This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/prot.24868 The crystal structure of the C-terminal domain of the Bacillus megaterium YpeB protein has been solved by X-ray crystallography to 1.80 ? resolution. The full-length protein is essential in stabilising the SleB cortex lytic enzyme in Bacillus spores, and may have a role in regulating SleB activity during spore germination. The YpeB-C crystal structure comprises three t...

  3. Identification of CdnL, a Putative Transcriptional Regulator Involved in Repair and Outgrowth of Heat-Damaged Bacillus cereus Spores.

    Directory of Open Access Journals (Sweden)

    Alicja K Warda

    Full Text Available Spores are widely present in the environment and are common contaminants in the food chain, creating a challenge for food industry. Nowadays, heat treatments conventionally applied in food processing may become milder to comply with consumer desire for products with higher sensory and nutritional values. Consequently subpopulations of spores may emerge that are sublethally damaged rather than inactivated. Such spores may germinate, repair damage, and eventually grow out leading to uncontrolled spoilage and safety issues. To gain insight into both the behaviour of damaged Bacillus cereus spores, and the process of damage repair, we assessed the germination and outgrowth performance using OD595 measurements and microscopy combined with genome-wide transcription analysis of untreated and heat-treated spores. The first two methods showed delayed germination and outgrowth of heat-damaged B. cereus ATCC14579 spores. A subset of genes uniquely expressed in heat-treated spores was identified with putative roles in the outgrowth of damaged spores, including cdnL (BC4714 encoding the putative transcriptional regulator CdnL. Next, a B. cereus ATCC14579 cdnL (BC4714 deletion mutant was constructed and assessment of outgrowth from heat-treated spores under food relevant conditions showed increased damage compared to wild type spores. The approach used in this study allows for identification of candidate genes involved in spore damage repair. Further identification of cellular parameters and characterisation of the molecular processes contributing to spore damage repair may provide leads for better control of spore outgrowth in foods.

  4. A procedure for estimating Bacillus cereus spores in soil and stream-sediment samples - A potential exploration technique

    Science.gov (United States)

    Watterson, J.R.

    1985-01-01

    The presence of bacterial spores of the Bacillus cereus group in soils and stream sediments appears to be a sensitive indicator of several types of concealed mineral deposits, including vein-type gold deposits. The B. cereus assay is rapid, inexpensive, and inherently reproducible. The test, currently under investigation for its potential in mineral exploration, is recommended for use on a research basis. Among the aerobic spore-forming bacilli, only B. cereus and closely related strains produce an opaque zone in egg-yolk emulsion agar. This characteristic, also known as the Nagler of lecitho-vitellin reaction, has long been used to rapidly indentify and estimate presumptive B. cereus. The test is here adapted to permit rapid estimation of B. cereus spores in soil and stream-sediment samples. Relative standard deviation was 10.3% on counts obtained from two 40-replicate pour-plate determinations. As many as 40 samples per day can be processed. Enough procedural detail is included to permit investigation of the test in conventional geochemical laboratories using standard microbiological safety precautions. ?? 1985.

  5. 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).

    Science.gov (United States)

    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.

  6. Measurements of DNA Damage and Repair in Bacillus anthracis Sterne Spores by UV Radiation

    Science.gov (United States)

    2014-09-18

    increase of water, protein motility begins and enzymatic activity is initiated. The outgrowth period of germination is the only step that contains...process [23]. 17 Figure 5. The spore germination process. Germination occurs in 2 main stages. The initiation/activation step involves the...with a red fluorescent protein was transformed into Ba Sterne cells prior. Following irradiation, germination media was added and the spores were

  7. Inactivation of Bacillus sporothermodurans spores by nisin and temperature studied by design of experiments in water and milk.

    Science.gov (United States)

    Aouadhi, Chedia; Rouissi, Zeineb; Mejri, Slah; Maaroufi, Abderrazak

    2014-04-01

    Spores of Bacillus sporothermodurans are known to be a contaminant of dairy products and to be extremely heat-resistant. A central composite experimental design with three factors using response surface methodology was used to evaluate the effect of nisin (50-150 UI/mL), temperature (80-100 °C), and temperature-holding time (10-20 min) on the inactivation of B. sporothermodurans LTIS27 spores in distilled water, in skim milk and in chocolate milk. The experimental values were shown to be significantly in good agreement with the values predicted by the quadratic equation since the adjusted determination coefficients (Radj(2)) were around 0.97. By analyzing the response surfaces plots, the inactivation was shown to be higher in distilled water than in skim milk under all the conditions tested. Five-log cycle reductions of B. sporothermodurans spores were obtained after a treatment at 95 °C for 12 min in presence of 125 UI of nisin/mL in distilled water or at 100 °C for 13 min in presence of 134 UI of nisin/mL in skim milk or at 100 °C for 15 min in presence of 135 UI of nisin/mL in chocolate milk. This study showed the efficiency of nisin (15-184 UI/mL) in combination with temperature (73-106 °C) to inactivate spores of B. sporothermodurans in milk.

  8. Detection of Bacillus anthracis spores in water using biosensors based on magnetostrictive microcantilever coated with phage

    Science.gov (United States)

    Fu, Liling; Li, Suiqiong; Zhang, Kewei; Cheng, Z.-Y.; Barbaree, J. M.

    2007-04-01

    Microcantilevers (MCs) as state-of-art sensor platforms have been widely investigated. We recently introduced a new type of MC, magnetostrictive microcantilever (MSMC), as high performance sensor platform. The MSMC is a remote/wireless sensor platform and exhibits a high quality merit factor in liquid. In this paper, a MSMC-based biosensor is developed for detecting B. anthracis spores in liquid, a potential biothreaten agent. The results demonstrated the advantages of MSMCs as a sensor platform. MSMCs with different sizes were fabricated and utilized in the experiments. The MSMCs were coated with the filamentous phage as a bio-recognition element to capture the B. anthracis spores. The phage-coated MSMCs as biosensors were exposed to cultures containing target spores with concentrations ranging from 5 * 10 4 spores/mL to 5 * 10 8 spores/mL. The resonance frequency of the MSMC sensors in cultures was monitored in a real-time manner. The results showed that for MSMCs of 2.8 mm * 1.0 mm * 35 μm and with 1.4 mm * 0.8 mm * 35 μm have a detection limit of 10 5 and 10 4 spores/mL, respectively.

  9. A Type III protein-RNA toxin-antitoxin system from Bacillus thuringiensis promotes plasmid retention during spore development.

    Science.gov (United States)

    Short, Francesca L; Monson, Rita E; Salmond, George P C

    2015-01-01

    Members of the Bacillus cereus sensu lato group of bacteria often contain multiple large plasmids, including those encoding virulence factors in B. anthracis. Bacillus species can develop into spores in response to stress. During sporulation the genomic content of the cell is heavily compressed, which could result in counterselection of extrachromosomal genomic elements, unless they have robust stabilization and segregation systems. Toxin-antitoxin (TA) systems are near-ubiquitous in prokaryotes and have multiple biological roles, including plasmid stabilization during vegetative growth. Here, we have shown that a Type III TA system, based on an RNA antitoxin and endoribonuclease toxin, from plasmid pAW63 in Bacillus thuringiensis serovar kurstaki HD-73 can dramatically promote plasmid retention in populations undergoing sporulation and germination, and we provide evidence that this occurs through the post-segregational killing of plasmid-free forespores. Our findings show how an extremely common genetic module can be used to ensure plasmid maintenance during stress-induced developmental transitions, with implications for plasmid dynamics in B. cereus s.l. bacteria.

  10. Influence of sporulation medium composition on transcription of ger operons and the germination response of spores of Bacillus cereus ATCC 14579

    NARCIS (Netherlands)

    Hornstra, L.M.; Vries, de Y.P.; Vos, de W.M.; Abee, T.

    2006-01-01

    Bacillus cereus ATCC 14579 endospores were produced in Y1 medium, a nutrient-rich, chemically defined sporulation medium, and in modified G medium, containing low amounts of nutrients. The average transcription level of the seven ger operons per cell was 3.5 times higher in Y1 medium, and the spores

  11. 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)

    NARCIS (Netherlands)

    Dybwad, M.; Laaken, A.L. van der; Blatny, J.M.; Paauw, A.

    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 desorptio

  12. Comparison of Bacillus atrophaeus spore viability following exposure to detonation of C4 and to deflagration of halogen-containing thermites

    Science.gov (United States)

    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.

  13. Comparison of Bacillus atrophaeus spore viability following exposure to detonation of C4 and to deflagration of halogen-containing thermites

    Energy Technology Data Exchange (ETDEWEB)

    Tringe, J. W.; Létant, S. E.; Dugan, L. C.; Levie, H. W.; Kuhl, A. L.; Murphy, G. A.; Alves, S. W.; Vandersall, K. S. [Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94550 (United States); Pantoya, M. L. [Mechanical Engineering Department, Texas Tech University, Lubbock, Texas 79409 (United States)

    2013-12-21

    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.

  14. Crystal structure of the PepSY-containing domain of the YpeB protein involved in germination of bacillus spores.

    Science.gov (United States)

    Üstok, Fatma Işık; Chirgadze, Dimitri Y; Christie, Graham

    2015-10-01

    The crystal structure of the C-terminal domain of the Bacillus megaterium YpeB protein has been solved by X-ray crystallography to 1.80-Å resolution. The full-length protein is essential in stabilising the SleB cortex lytic enzyme in Bacillus spores, and may have a role in regulating SleB activity during spore germination. The YpeB-C crystal structure comprises three tandemly repeated PepSY domains, which are aligned to form an extended laterally compressed molecule. A predominantly positively charged region located in the second PepSY domain may provide a site for protein interactions that are important in stabilising SleB and YpeB within the spore.

  15. Spore Resistance Properties.

    Science.gov (United States)

    Setlow, Peter

    2014-10-01

    Spores of various Bacillus and Clostridium species are among the most resistant life forms known. Since the spores of some species are causative agents of much food spoilage, food poisoning, and human disease, and the spores of Bacillus anthracis are a major bioweapon, there is much interest in the mechanisms of spore resistance and how these spores can be killed. This article will discuss the factors involved in spore resistance to agents such as wet and dry heat, desiccation, UV and γ-radiation, enzymes that hydrolyze bacterial cell walls, and a variety of toxic chemicals, including genotoxic agents, oxidizing agents, aldehydes, acid, and alkali. These resistance factors include the outer layers of the spore, such as the thick proteinaceous coat that detoxifies reactive chemicals; the relatively impermeable inner spore membrane that restricts access of toxic chemicals to the spore core containing the spore's DNA and most enzymes; the low water content and high level of dipicolinic acid in the spore core that protect core macromolecules from the effects of heat and desiccation; the saturation of spore DNA with a novel group of proteins that protect the DNA against heat, genotoxic chemicals, and radiation; and the repair of radiation damage to DNA when spores germinate and return to life. Despite their extreme resistance, spores can be killed, including by damage to DNA, crucial spore proteins, the spore's inner membrane, and one or more components of the spore germination apparatus.

  16. Purine and its analogues and radiation damage in Bacillus megaterium spores

    Energy Technology Data Exchange (ETDEWEB)

    Powers, E.L.

    1986-12-01

    As an extension of results obtained from radiation studies on caffeine both in other laboratories and more recently in this laboratory using the bacterial spore as the test system, six compounds with chemical structures closely resembling that of caffeine were tested as radiation modifiers. Of these compounds, purine, adenine and hypoxanthine resembled caffeine in sensitizing spores to radiation, while theobromine, xanthine and theophylline did not. These responses are discussed in relation to the electron sequestration hypothesis of cellular sensitization to high-energy radiation.

  17. Decontamination of Bacillus spores adhered to iron and cement-mortar drinking water infrastructure in a model system using disinfectants.

    Science.gov (United States)

    Szabo, Jeffrey G; Meiners, Greg; Heckman, Lee; Rice, Eugene W; Hall, John

    2017-02-01

    Decontamination of Bacillus spores adhered to common drinking water infrastructure surfaces was evaluated using a variety of disinfectants. Corroded iron and cement-mortar lined iron represented the infrastructure surfaces, and were conditioned in a 23 m long, 15 cm diameter (75 ft long, 6 in diameter) pilot-scale drinking water distribution pipe system. Decontamination was evaluated using increased water velocity (flushing) alone at 0.5 m s(-1) (1.7 ft s(-1)), as well as free chlorine (5 and 25 mg L(-1)), monochloramine (25 mg L(-1)), chlorine dioxide (5 and 25 mg L(-1)), ozone (2.0 mg L(-1)), peracetic acid 25 mg L(-1)) and acidified nitrite (0.1 mol L(-1) at pH 2 and 3), all followed by flushing at 0.3 m s(-1) (1 ft s(-1)). Flushing alone reduced the adhered spores by 0.5 and 2.0 log10 from iron and cement-mortar, respectively. Log10 reduction on corroded iron pipe wall coupons ranged from 1.0 to 2.9 at respective chlorine dioxide concentrations of 5 and 25 mg L(-1), although spores were undetectable on the iron surface during disinfection at 25 mg L(-1). Acidified nitrite (pH 2, 0.1 mol L(-1)) yielded no detectable spores on the iron surface during the flushing phase after disinfection. Chlorine dioxide was the best performing disinfectant with >3.0 log10 removal from cement-mortar at 5 and 25 mg L(-1). The data show that free chlorine, monochloramine, ozone and chlorine dioxide followed by flushing can reduce adhered spores by > 3.0 log10 on cement-mortar.

  18. Fate of pathogenic Bacillus cereus spores after ingestion by protist grazers

    DEFF Research Database (Denmark)

    Winding, Anne; Santos, Susana; Hendriksen, Niels Bohse

    was initially investigated in microcosms inoculated with pure cultures of the protists Acanthamoeba castellanii, Tetrahymena pyriformis and Cercomonas sp. as grazers. Individual protist cultures were fed with fluorescently labelled (CellTracker™RedCMTPX) B. cereus spores or vegetative cells as the only food...

  19. Removal of Bacillus anthracis sterne spore from commercial unpasteurized liquid egg white

    Science.gov (United States)

    Thermal pasteurization used by the egg industry for controlling vegetative cells of pathogens is ineffective for destroying endospores. There is a strong need in the agri-industries to develop effective intervention strategies to eliminate the possible bioterrorism threat from spore forming bacteria...

  20. Decontamination of Streptococci biofilms and Bacillus cereus spores on plastic surfaces with DC and pulsed corona discharges

    Science.gov (United States)

    Koval'ová, Zuzana; Tarabová, Kataŕna; Hensel, Karol; Machala, Zdenko

    2013-02-01

    Cold air plasmas of DC and pulsed corona discharges: positive streamers and negative Trichel pulses were used for bio-decontamination of Streptococci biofilm and Bacillus cereus spores on polypropylene plastic surfaces. The reduction of bacterial population (evaluated as log10) in the biofilm on plastic surfaces treated by DC corona reached 2.4 logs with 10 min treatment time and 3.3 logs with 2 min treatment time with water spraying. The enhancement of plasma biocidal effects on the biofilm by electro-spraying of water through a hollow needle high-voltage electrode was investigated. No significant polarity effect was found with DC corona. Pulsed corona was demonstrated slightly more bactericidal for spores, especially in the negative polarity where the bacterial population reduction reached up to 2.2 logs at 10 min exposure time. Contribution to the Topical Issue "13th International Symposium on High Pressure Low Temperature Plasma Chemistry (Hakone XIII)", Edited by Nicolas Gherardi, Henryca Danuta Stryczewska and Yvan Ségui.

  1. Synergistic effect of sequential or combined use of ozone and UV radiation for the disinfection of Bacillus subtilis spores.

    Science.gov (United States)

    Jung, Yeon Jung; Oh, Byung Soo; Kang, Joon-Wun

    2008-03-01

    This study was performed to evaluate the inactivation efficiency or synergy of combined ozone and UV processes (combined ozone/UV process) or sequential processes (ozone-UV, UV-ozone) compared with individual unit processes and to investigate the specific roles of ozone, UV and the hydroxyl radical, which is formed as an intermediate in the combined ozone/UV process. The Bacillus subtilis spore, which has often been used as a surrogate microorganism for Cryptosporidium parvum oocysts, was used as a target microorganism. Compared to individual unit processes with ozone or UV, the inactivation of B. subtilis spores by the combined ozone/UV process was enhanced under identical conditions. To investigate the specific roles of ozone and UV in the combined ozone/UV process, sequential ozone-UV and UV-ozone processes were tested for degrees of inactivation. Additionally, the experiment was performed in the presence and absence of tert-butyl alcohol, which acted as a hydroxyl radical scavenger to assess the role of inactivation by the hydroxyl radical in the combined ozone/UV process. Among the five candidate processes, the greatest synergistic effect was observed in the combined ozone/UV process. From the comparison of five candidate processes, the hydroxyl radical and ozone were each determined to significantly enhance the overall inactivation efficiency in the combined ozone/UV process.

  2. Activity of spores and extracellular proteins from six Cry+ strains and a Cry- strain of Bacillus thuringiensis subsp. kurstaki against the western spruce budworm, Choristoneura occidentalis (Lepidoptera: Tortricidae).

    Science.gov (United States)

    Kalmykova, Galina; Burtseva, Ljudmila; Milne, Ross; van Frankenhuyzen, Kees

    2009-05-01

    We characterized insecticidal activity of previously untested strains of Bacillus thuringiensis kurstaki belonging to two crystal serovars (K-1 and K-73) against the western spruce budworm (Choristoneura occidentalis Freeman 1967). By testing various components, we demonstrated that spores play a critical role in the pathogenesis of each strain. Spore-free crystals caused low mortality and purified spores were generally not toxic. The addition of spores to purified protoxin increased toxicity several hundred-fold, regardless of the parental strain from which the spores or protoxins were derived. The crystal and spore components did not account for full insecticidal activity of whole sporulated cultures owing to the toxicity of soluble proteins that are secreted during cell growth. We observed a marked difference in toxicity of secreted proteins between the K-1 and K-73 type strains, with the K-1 preparations causing much higher mortality, mass reduction, and inhibition of pupation. There was a consistent correlation between relative toxicity of secreted protein preparations and the presence and quantity of the Vip3A protein, suggesting that this protein contributes to the virulence of B. thuringiensis subsp. kurstaki in western spruce budworm larvae. However, other virulence factors have to be invoked to explain the synergizing effect of spores from both K-1 and K-73 strains on Cry protein toxicity.

  3. Discrimination of Bacillus anthracis Spores by Direct in-situ Analysis of Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Youngsu; Lee, Jonghee; Kim, Seongsoo [Agency for Defense Development, Daejeon (Korea, Republic of)

    2013-09-15

    The rapid and accurate identification of biological agents is a critical step in the case of bio-terror and biological warfare attacks. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been widely used for the identification of microorganisms. In this study, we describe a method for the rapid and accurate discrimination of Bacillus anthracis spores using MALDI-TOF MS. Our direct in-situ analysis of MALDI-TOF MS does not involve subsequent high-resolution mass analyses and sample preparation steps. This method allowed the detection of species-specific biomarkers from each Bacillus spores. Especially, B. anthracis spores had specific biomarker peaks at 2503, 3089, 3376, 6684, 6698, 6753, and 6840 m/z. Cluster and PCA analyses of the mass spectra of Bacillus spores revealed distinctively separated clusters and within-groups similarity. Therefore, we believe that this method is effective in the real-time identification of biological warfare agents such as B. anthracis as well as other microorganisms in the field.

  4. National validation study of a swab protocol for the recovery of Bacillus anthracis spores from surfaces.

    Science.gov (United States)

    Hodges, Lisa R; Rose, Laura J; O'Connell, Heather; Arduino, Matthew J

    2010-05-01

    Twelve Laboratory Response Network (LRN) affiliated laboratories participated in a validation study of a macrofoam swab protocol for the recovery, detection, and quantification of viable B. anthracis (BA) Sterne spores from steel surfaces. CDC personnel inoculated steel coupons (26cm(2)) with 1-4 log(10) BA spores and recovered them by sampling with pre-moistened macrofoam swabs. Phase 1 (P1) of the study evaluated swabs containing BA only, while dust and background organisms were added to swabs in Phase 2 (P2) to mimic environmental conditions. Laboratories processed swabs and enumerated spores by culturing eluted swab suspensions and counting colonies with morphology consistent with BA. Processed swabs were placed in enrichment broth, incubated 24h, and cultured by streaking for isolation. Real-time PCR was performed on selected colonies from P2 samples to confirm the identity of BA. Mean percent recovery (%R) of spores from the surface ranged from 15.8 to 31.0% (P1) and from 27.9 to 55.0% (P2). The highest mean percent recovery was 31.0% (sd 10.9%) for P1 (4 log(10) inoculum) and 55.0% (sd 27.6%) for P2 (1 log(10) inoculum). The overall %R was higher for P2 (44.6%) than P1 (24.1%), but the overall reproducibility (between-lab variability) was lower in P2 than in P1 (25.0 vs 16.5%CV, respectively). The overall precision (within-lab variability) was close to identical for P1 and P2 (44.0 and 44.1, respectively), but varied greatly between inoculum levels. The protocol demonstrated linearity in %R over the three inoculum levels and is able to detect between 26 and 5x10(6)spores/26cm(2). Sensitivity as determined by culture was >98.3% for both phases and all inocula, suggesting that the culture method maintains sensitivity in the presence of contaminants. The enrichment broth method alone was less sensitive for sampled swabs (66.4%) during P2, suggesting that the presence of background organisms inhibited growth or isolation of BA from the broth. The addition of

  5. Effects of two probiotic additives containing Bacillus spores on carcass characteristics, blood lipids and cecal volatile fatty acids in meat type chickens.

    Science.gov (United States)

    Novak, R; Bogovič Matijašić, B; Terčič, D; Cervek, M; Gorjanc, G; Holcman, A; Levart, A; Rogelj, I

    2011-08-01

    The objective of this study was to evaluate effects of two commercially available probiotic additives, containing Bacillus spores, on carcass and meat characteristics, serum lipids and concentration of cecal volatile fatty acids of meat type chickens. Birds were fed regular corn-soy meal based feed (control), supplemented with additive A, containing 1.6 × 10(6) spores per gram of feed of Bacillus subtilis and Bacillus licheniformis (group A) or additive B, containing the same concentration of Bacillus cereus var. toyoi spores (group B). One hundred and twenty birds (20 per replicate) were slaughtered at the age of 55 days. Results showed that birds in group B had higher (p blood serum cholesterol profile. Both probiotics influenced the cecal fermentation, which was observed as decrease in cecal concentrations of propionic, butyric, n-butyric and n-valeric acids, but the differences compared to control group were statistically significant for group A only. It was established that probiotic additive B was more effective regarding carcass and meat part weights than additive A, however the animals from group B also had more abdominal fat and their meat had significantly higher conductivity than control group, which is not considered as beneficial.

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

    Science.gov (United States)

    2009-12-01

    Fritsch, and T. Maniatis . 1989. Molecular cloning : a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 37...on ExsFA/BxpB. In spores lacking the exosporium surface protein BclA, ExsK fails to mature into high- molecular -mass species observed in wild-type...the envi- ronment. To gain insight into the molecular basis of exosporium as- sembly and function, we studied a previously identified but otherwise

  7. Improved Proteomic Analysis Following Trichloroacetic Acid Extraction of Bacillus anthracis Spore Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kaiser, Brooke LD; Wunschel, David S.; Sydor, Michael A.; Warner, Marvin G.; Wahl, Karen L.; Hutchison, Janine R.

    2015-08-07

    Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Proteomic analysis is dependent upon efficient extraction of proteins from bacterial samples without introducing bias toward extraction of particular protein classes. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrich for certain classes of proteins. The method presented here is technically simple and does not require specialized equipment such as a mechanical disrupter. Our data reveal that for particularly challenging samples, such as B. anthracis Sterne spores, trichloroacetic acid extraction improved the number of proteins identified within a sample compared to bead beating (714 vs 660, respectively). Further, TCA extraction enriched for 103 known spore specific proteins whereas bead beating resulted in 49 unique proteins. Analysis of C. botulinum samples grown to 5 days, composed of vegetative biomass and spores, showed a similar trend with improved protein yields and identification using our method compared to bead beating. Interestingly, easily lysed samples, such as B. anthracis vegetative cells, were equally as effectively processed via TCA and bead beating, but TCA extraction remains the easiest and most cost effective option. As with all assays, supplemental methods such as implementation of an alternative preparation method may provide additional insight to the protein biology of the bacteria being studied.

  8. Deletion in sigB in Bacillus cereus affects spore properties

    NARCIS (Netherlands)

    Vries, de Y.P.; Hornstra, L.M.; Atmadja, R.D.; Schaik, van W.; Vos, de W.M.; Abee, T.

    2005-01-01

    In Bacillus cereus and other gram-positive bacteria the alternative sigma factor ¿B is an important regulator of the stress response. Deletion of the sigB gene generally leads to a stress-sensitive phenotype of vegetative cells. In this study, we describe the effect of the deletion of the sigB gene

  9. Ultra high pressure homogenization (UHPH inactivation of Bacillus amyloliquefaciens spores in phosphate buffered saline (PBS and milk

    Directory of Open Access Journals (Sweden)

    Peng eDong

    2015-07-01

    Full Text Available Ultra high pressure homogenization (UHPH opens up new areas for dynamic high pressure assisted thermal sterilization of liquids. Bacillus amyloliquefaciens spores are resistant to high isostatic pressure and temperature and were suggested as potential surrogate for high pressure thermal sterilization validation. B. amyloliquefaciens spores suspended in PBS buffer (0.01 M, pH 7.0, low fat milk (1.5%, pH 6.7 and whole milk (3.5%, pH 6.7 at initial concentration of ~106 CFU/mL were subjected to UHPH treatments at 200, 300 and 350 MPa with an inlet temperature at ~80 °C. Thermal inactivation kinetics of B. amyloliquefaciens spores in PBS and milk were assessed with thin wall glass capillaries and modeled using mechanistic linear first order and Weibull models. The residence time during UHPH treatments was estimated to determine the contribution of temperature to spore inactivation by UHPH. No sublethal injury was detected after UHPH treatments using sodium chloride as selective component in the nutrient agar medium. The inactivation profiles of spores in PBS buffer and milk were compared and fat provided no clear protective effect for spores against treatments. Treatment at 200 MPa with valve temperatures lower than 125 °C caused no reduction of spores. A reduction of 3.5 log10 CFU/mL of B. amyloliquefaciens spores was achieved by treatment at 350 MPa with a valve temperature higher than 150 °C. The modeled thermal inactivation and observed inactivation during UHPH treatments suggest that temperature could be the main lethal effect driving inactivation.

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

    Science.gov (United States)

    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.

  11. Comparison of eight methods for the extraction of Bacillus atrophaeus spore DNA from eleven common interferents and a common swab.

    Directory of Open Access Journals (Sweden)

    Helen L Rose

    Full Text Available Eight DNA extraction products or methods (Applied Biosystems PrepFiler Forensic DNA Extraction Kit; Bio-Rad Instagene Only, Bio-Rad Instagene & Spin Column Purification; EpiCentre MasterPure DNA & RNA Kit; FujiFilm QuickGene Mini80; Idaho Technologies 1-2-3 Q-Flow Kit; MoBio UltraClean Microbial DNA Isolation Kit; Sigma Extract-N-Amp Plant and Seed Kit were adapted to facilitate extraction of DNA under BSL3 containment conditions. DNA was extracted from 12 common interferents or sample types, spiked with spores of Bacillus atropheaus. Resulting extracts were tested by real-time PCR. No one method was the best, in terms of DNA extraction, across all sample types. Statistical analysis indicated that the PrepFiler method was the best method from six dry powders (baking, biological washing, milk, plain flour, filler and talcum and one solid (Underarm deodorant, the UltraClean method was the best from four liquids (aftershave, cola, nutrient broth, vinegar, and the MasterPure method was the best from the swab sample type. The best overall method, in terms of DNA extraction, across all sample types evaluated was the UltraClean method.

  12. Chronic toxicity and physiological changes induced in the honey bee by the exposure to fipronil and Bacillus thuringiensis spores alone or combined.

    Science.gov (United States)

    Renzi, Maria Teresa; Amichot, Marcel; Pauron, David; Tchamitchian, Sylvie; Brunet, Jean-Luc; Kretzschmar, André; Maini, Stefano; Belzunces, Luc P

    2016-05-01

    In the agricultural environment, honey bees may be exposed to combinations of pesticides. Until now, the effects of these combinations on honey bee health have been poorly investigated. In this study, we assessed the impacts of biological and chemical insecticides, combining low dietary concentrations of Bacillus thuringiensis (Bt) spores (100 and 1000µg/L) with the chemical insecticide fipronil (1µg/L). In order to assess the possible effects of Cry toxins, the Bt kurstaki strain (Btk) was compared with a Bt strain devoid of toxin-encoding plasmids (Bt Cry(-)). The oral exposure to fipronil and Bt spores from both strains for 10 days did not elicit significant effects on the feeding behavior and survival after 25 days. Local and systemic physiological effects were investigated by measuring the activities of enzymes involved in the intermediary and detoxication metabolisms at two sampling dates (day 10 and day 20). Attention was focused on head and midgut glutathione-S-transferase (GST), midgut alkaline phosphatase (ALP), abdomen glyceraldehyde-3-phosphate dehydrogenase (GAPD) and glucose-6-phosphate dehydrogenase (G6PD). We found that Bt Cry(-) and Btk spores induced physiological modifications by differentially modulating enzyme activities. Fipronil influenced the enzyme activities differently at days 10 and 20 and, when combined with Bt spores, elicited modulations of some spore-induced physiological responses. These results show that an apparent absence of toxicity may hide physiological disruptions that could be potentially damaging for the bees, especially in the case of combined exposures to other environmental stressors.

  13. Comprehensive Laboratory Evaluation of a Highly Specific Lateral Flow Assay for the Presumptive Identification of Bacillus anthracis Spores in Suspicious White Powders and Environmental Samples

    Science.gov (United States)

    Ramage, Jason G.; Prentice, Kristin W.; DePalma, Lindsay; Venkateswaran, Kodumudi S.; Chivukula, Sruti; Chapman, Carol; Bell, Melissa; Datta, Shomik; Singh, Ajay; Hoffmaster, Alex; Sarwar, Jawad; Parameswaran, Nishanth; Joshi, Mrinmayi; Thirunavkkarasu, Nagarajan; Krishnan, Viswanathan; Morse, Stephen; Avila, Julie R.; Sharma, Shashi; Estacio, Peter L.; Stanker, Larry; Hodge, David R.

    2016-01-01

    We conducted a comprehensive, multiphase laboratory evaluation of the Anthrax BioThreat Alert® test strip, a lateral flow immunoassay (LFA) for the rapid detection of Bacillus anthracis spores. The study, conducted at 2 sites, evaluated this assay for the detection of spores from the Ames and Sterne strains of B. anthracis, as well as those from an additional 22 strains. Phylogenetic near neighbors, environmental background organisms, white powders, and environmental samples were also tested. The Anthrax LFA demonstrated a limit of detection of about 106 spores/mL (ca. 1.5 × 105 spores/assay). In this study, overall sensitivity of the LFA was 99.3%, and the specificity was 98.6%. The results indicated that the specificity, sensitivity, limit of detection, dynamic range, and repeatability of the assay support its use in the field for the purpose of qualitatively evaluating suspicious white powders and environmental samples for the presumptive presence of B. anthracis spores. PMID:27661796

  14. Protection of Penaeus monodon against white spot syndrome by continuous oral administration of a low concentration of Bacillus subtilis spores expressing the VP28 antigen.

    Science.gov (United States)

    Pham, K-C; Tran, H T T; Van Doan, C; Le, P H; Van Nguyen, A T; Nguyen, H A; Hong, H A; Cutting, S M; Phan, T-N

    2017-03-01

    In this study, Bacillus subtilis spores expressing a chimeric protein, CotB-VP28, were used as a probiotic vaccine to protect black tiger shrimps (Penaeus monodon) against white spot syndrome virus (WSSV) infection. Oral administration of pellets coated with CotB-VP28 spores (at ≥1 × 10(9 ) CFU per g pellet) to shrimps induced immune-relating phenoloxydase activity (PO) in shrimps after 14 days of feeding (prior challenge) and at day 3 post challenge (1·26 and 1·70 fold increase respectively). A 75% protection rate was obtained by continuous feeding of the spore-coated pellets at ≥1 × 10(9 ) CFU per g for 14 days prior to WSSV challenge and during all the postchallenge period. Even when the amount of CotB-VP28 spores in feed pellets was reduced down to ≥5 × 10(7)  CFU per g and ≥1 × 10(6)  CFU per g, relatively high protection rates of 70 and 67·5%, respectively, were still obtained. By contrast, feeding pellets without spores (untreated group) and with naked spores (PY79 group) at ≥1 × 10(9)  CFU per g could not protect shrimps against WSSV. These data suggest that supplementation of CotB-VP28 spores at low dose of ≥1 × 10(6)  CFU per g could be effective as a prophylactic treatment of WSS for black tiger shrimps.

  15. Recombinant Bacillus subtilis spores expressing cholera toxin B subunit and Helicobacter pylori urease B confer protection against H. pylori in mice.

    Science.gov (United States)

    Zhou, Zhenwen; Dong, Hui; Huang, Yanmei; Yao, Shuwen; Liang, Bingshao; Xie, Yongqiang; Long, Yan; Mai, Jialiang; Gong, Sitang

    2017-01-01

    Helicobacter pylori infection is associated with chronic gastritis, peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue lymphoma. The limitations of current therapies for H. pylori infection include poor compliance and antibiotic resistance. Therefore, an effective anti-H. pylori vaccine would be an alternative or complement to antibiotic treatment. Urease B (UreB) is considered an ideal vaccine antigen against H. pylori infection. In this study, cholera toxin B subunit (CTB), a mucosal adjuvant, was used to enhance the immunogenicity of a novel Bacillus subtilis spore vaccine expressing CTB-UreB, along with the B. subtilis spore coat protein CotC as a fusion protein. Oral administration of B. subtilis spores expressing CotC-UreB or CotC-CTB-UreB led to increased levels of UreB-specific IgG in serum and UreB-specific IgA in faeces, as well as elevated levels of IL-10 and IFN-γ in splenocytes. In addition, oral administration of CotC-UreB or CotC-CTB-UreB spores induced significant reductions (80.0 and 90.5 %, respectively) in gastric H. pylori bacterial load (1.11±0.36×105 and 0.53±0.21×105 c.f.u., respectively) compared to that of the CotC control group (5.56±1.64×105 c.f.u., P<0.01). Moreover, CotC-CTB-UreB spores were significantly more effective at reducing the bacterial load than CotC-UreB spores (P<0.05). These results indicate that CotC-CTB-UreB-expressing B. subtilis spores are a potential vaccine candidate for the control of H. pylori infection.

  16. Development of a powder formulation based on Bacillus cereus sensu lato strain B25 spores for biological control of Fusarium verticillioides in maize plants.

    Science.gov (United States)

    Martínez-Álvarez, Juan C; Castro-Martínez, Claudia; Sánchez-Peña, Pedro; Gutiérrez-Dorado, Roberto; Maldonado-Mendoza, Ignacio E

    2016-05-01

    Maize is an economically important crop in northern Mexico. Different fungi cause ear and root rot in maize, including Fusarium verticillioides (Sacc.) Nirenberg. Crop management of this pathogen with chemical fungicides has been difficult. By contrast, the recent use of novel biocontrol strategies, such as seed bacterization with Bacillus cereus sensu lato strain B25, has been effective in field trials. These approaches are not without their problems, since insufficient formulation technology, between other factors, can limit success of biocontrol agents. In response to these drawbacks, we have developed a powder formulation based on Bacillus B25 spores and evaluated some of its characteristics, including shelf life and efficacy against F. verticillioides, in vitro and in maize plants. A talc-based powder formulation containing 1 × 10(9) c.f.u. g(-1) was obtained and evaluated for seed adherence ability, seed germination effect, shelf life and antagonism against F. verticillioides in in vitro and in planta assays. Seed adherence of viable bacterial spores ranged from 1.0 to 1.41 × 10(7) c.f.u. g(-1). Bacteria did not display negative effects on seed germination. Spore viability for the powder formulation slowly decreased over time, and was 53 % after 360 days of storage at room temperature. This formulation was capable of controlling F. verticillioides in greenhouse assays, as well as eight other maize phytopathogenic fungi in vitro. The results suggest that a talc-based powder formulation of Bacillus B25 spores may be sufficient to produce inoculum for biocontrol of maize ear and root rots caused by F. verticillioides.

  17. Site-Directed Mutagenesis and Structural Studies Suggest that the Germination Protease, GPR, in Spores of Bacillus Species Is an Atypical Aspartic Acid Protease

    Science.gov (United States)

    Carroll, Thomas M.; Setlow, Peter

    2005-01-01

    Germination protease (GPR) initiates the degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus and Clostridium species. The GPR amino acid sequence is not homologous to members of the major protease families, and previous work has not identified residues involved in GPR catalysis. The current work has focused on identifying catalytically essential amino acids by mutagenesis of Bacillus megaterium gpr. A residue was selected for alteration if it (i) was conserved among spore-forming bacteria, (ii) was a potential nucleophile, and (iii) had not been ruled out as inessential for catalysis. GPR variants were overexpressed in Escherichia coli, and the active form (P41) was assayed for activity against SASP and the zymogen form (P46) was assayed for the ability to autoprocess to P41. Variants inactive against SASP and unable to autoprocess were analyzed by circular dichroism spectroscopy and multiangle laser light scattering to determine whether the variant's inactivity was due to loss of secondary or quaternary structure, respectively. Variation of D127 and D193, but no other residues, resulted in inactive P46 and P41, while variants of each form were well structured and tetrameric, suggesting that D127 and D193 are essential for activity and autoprocessing. Mapping these two aspartate residues and a highly conserved lysine onto the B. megaterium P46 crystal structure revealed a striking similarity to the catalytic residues and propeptide lysine of aspartic acid proteases. These data indicate that GPR is an atypical aspartic acid protease. PMID:16199582

  18. In situ detection of Bacillus anthracis spores using fully submersible, self-exciting, self-sensing PMN-PT/Sn piezoelectric microcantilevers.

    Science.gov (United States)

    McGovern, John-Paul; Shih, Wan Y; Shih, Wei-Heng

    2007-08-01

    In this study, we have demonstrated in situ, all-electrical detection of Bacillus anthracis (BA) spores using lead magnesium niobate-lead titanate/tin (PMN-PT/Sn) piezoelectric microcantilever sensors (PEMS) fabricated from PMN-PT freestanding films and electrically insulated with methyltrimethoxysilane (MTMS) coatings on the tin surface. Antibody specific to BA spore surface antigen was immobilized on the platinum electrode of the PMN-PT layer. In phosphate-buffered saline (PBS) solution, the PMN-PT/Sn PEMS exhibited quality (Q) values ranging from 50 to 75. The detection was carried out in a closed-loop flow cell with a liquid volume of 0.8 ml and a flow rate of 1 ml min(-1). It was shown that one sensor, "PEMS-A" (500 microm long, 800 microm wide, with a 22 microm thick PMN-PT layer, a 20 microm thick tin layer and a 1 +/- 0.5 x 10(-12) g Hz(-1) mass detection sensitivity) exhibited resonance frequency shifts of 2100 +/- 200, 1100 +/- 100 and 700 +/- 100 Hz at concentrations of 20,000, 2000, and 200 spores ml(-1) or 16,000, 1600, and 160 total spores, respectively. Additionally, "PEMS-B" (350 microm long, 800 microm wide, with an 8 microm thick PMN-PT layer, a 6 microm thick tin layer and a 2 +/- 1 x 10(-13) g Hz(-1) mass detection sensitivity) exhibited resonance frequency shifts of 2400 +/- 200, 1500 +/- 200, 500 +/- 150 and 200 +/- 100 Hz at concentrations of 20,000, 2000, 100, and 45 spores ml(-1) or 16,000, 1600, 80, and 36 total spores, respectively.

  19. Comparative evaluation of eleven commercial DNA extraction kits for real-time PCR detection of Bacillus anthracis spores in spiked dairy samples.

    Science.gov (United States)

    Mertens, Katja; Freund, Lisa; Schmoock, Gernot; Hänsel, Christoph; Melzer, Falk; Elschner, Mandy C

    2014-01-17

    Spores of Bacillus anthracis are highly resistant and can survive conditions used for food preservation. Sample size and complexity represent the major hurdles for pathogen detection in food-related settings. Eleven commercial DNA extraction kits were evaluated for detection of B. anthracis spores by quantitative real-time PCR (qPCR) in dairy products. DNA was extracted from serial dilutions of B. anthracis spores in milk powder, cream cheese, whole milk and buttermilk. Three kits (QIAamp DNA mini kit, Invisorb Food kit I and II) were determined to produce the lowest limit of detections (LODs) with equally good performance. These kits employed lysozyme and proteinase K treatments or proteinase K in combination with cethyltrimethylamonium bromide-mediated (CTAB) precipitation of cell debris for cell disruption and DNA release. The LODs for these three kits were determined as 10(2) spores/ml of distilled water, 10(3)s pores/20 mg of powdered milk and 10(4) spores/100 mg of cream cheese, respectively. Performance testing of the QIAamp DNA mini kit demonstrated a good reproducibility and appropriate detection limits from 10(3)/ml for butter milk, 10(4)/ml for whole milk and 10(4)/100 mg for low fat cream cheese. However, DNA extraction efficiency was strongly inhibited by cream cheese with higher fat contents with an increased LOD of 10(6)/100 mg spores. This study demonstrated that qPCR detection depends directly on the appropriate DNA extraction method for an individual food matrix and bacterial agent.

  20. Spore stage expression of a vegetative insecticidal gene increase toxicity of Bacillus thuringiensis subsp. aizawai SP41 against Spodoptera exigua.

    Science.gov (United States)

    Thamthiankul Chankhamhaengdecha, S; Tantichodok, A; Panbangred, Watanalai

    2008-09-10

    To enhance the toxicity of the Bacillus thuringiensis subsp. aizawai strain SP41 (SP41), the vegetative insecticidal protein (Vip) gene vip3A from SP41 was redirected to the sporulation stage by replacing its native promoter with the strong promoter P19 of the cry11Aa operon. Compared to the wild type, SP41 with PVIP (vip3A with its native promoter and ter) had the relative expression ratios of 457, 548, and 290 at 8, 14, and 20 h of cultivation, respectively, as measured by quantitative reverse transcription polymerase chain reaction (PCR). SP41 transformed by P19VIP (vip3A controlled by P19 promoter with vip3A ter) showed higher expressions (23, 2055, 1831) at the same time points. SP41 with P19VIP20 (vip3A controlled by P19 promoter and containing P20 and operon ter) had the lowest expression levels (3, 11, 9) at any time point. SDS-PAGE analysis of proteins in the culture supernatant of the P19VIP at 8, 14, and 20 h demonstrated a significant increase in Vip3A at the sporulation stage. Using the surface contamination bioassay, the 50% lethal concentration (LC(50)) of whole culture of PVIP, P19VIP, and P19VIP20 at 20 and 48 h of cultivation against Spodoptera exigua larvae were (68.3, 21.2, and 60.2 microg cm(-2)) and (69.8, 41.8, and 74.6 microg cm(-2)), respectively, compared with 86.6 and 104.4 microg cm(-2) for SP41. The results showed that Vip from P19VIP, expressed at spore stage at 20 and 48 h, can increase the toxicity of SP41 for 4.1- and 2.5-fold, respectively.

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

    Science.gov (United States)

    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.

  2. Comparative analysis of the immunologic response induced by the Sterne 34F2 live spore Bacillus anthracis vaccine in a ruminant model.

    Science.gov (United States)

    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.

  3. Fate and effect of ingested Bacillus cereus spores and vegetative cells in the intestinal tract of human-flora-associated rats

    DEFF Research Database (Denmark)

    Wilcks, Andrea; Hansen, Bjarne Munk; Hendriksen, Niels Bohse;

    2006-01-01

    The fate and effect of Bacillus cereus F4433/73R in the intestine of human-flora-associated rats was studied using bacteriological culturing techniques and PCR-denaturing gradient gel electrophoresis in combination with cell assays and immunoassays for detection of enterotoxins. In faecal samples...... gradient gel electrophoresis analysis with universal 16S rRNA gene primers revealed significant changes in the intestinal microbiota of animals dosed with spores. Vero cell assays and a commercial kit (BCET-RPLA) did not reveal any enterotoxin production from B. cereus F4433/73R in the intestinal tract....

  4. Inactivation of Bacillus anthracis spores by a combination of biocides and heating under high-temperature short-time pasteurization conditions.

    Science.gov (United States)

    Xu, Sa; Labuza, Theodore P; Diez-Gonzalez, Francisco

    2008-06-01

    The milk supply is considered a primary route for a bioterrorism attack with Bacillus anthracis spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate B. anthracis spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85 degrees C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80 degrees C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80 degrees C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80 degrees C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80 degrees C, respectively. TTI6-log of less than 20 s were also achieved at 80 degrees C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of B. anthracis spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be

  5. Anthrax Spores under a microscope

    Science.gov (United States)

    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.

  6. Inhibitory effects of nisin and potassium sorbate alone or in combination on vegetative cells growth and spore germination of Bacillus sporothermodurans in milk.

    Science.gov (United States)

    Aouadhi, Chedia; Mejri, Slah; Maaroufi, Abderrazak

    2015-04-01

    The inhibitory activities of nisin or/and potassium sorbate on spores and vegetative cells of Bacillus sporothermodurans LTIS27, which are known to be a contaminant of dairy products and to be extremely heat-resistant, were investigated. First, the tested concentrations of nisin or potassium sorbate inhibited vegetative cell growth; with the minimum inhibitory concentrations were 5 × 10(3) IU/ml and 2% (w/v), respectively. Then, the behaviour of vegetative cells and spores in presence of sub-lethal concentrations of nisin (50 UI/ml) or/and potassium sorbate (0.2%), in milk at 37 °C for 5 days, were evaluated. In the absence of inhibitors, strain grew and sporulated at the end of the exponential phase. Nisin (50 UI/ml) was able to inhibit spore outgrowth but didn't affect their germination. It induced an immediate and transitory reduction (1.6log(10) after 1 h and 2.8log(10) after 6 h of incubation) of vegetative cell growth which reappeared between 10 h and 24 h. Potassium sorbate (0.2%) had a durable bacteriostatic effect (1.1log(10) after 6 h), on vegetative cells, followed by a slower regrowth. It was able to inhibit both germination and outgrowth of spores. Association of nisin and potassium sorbate, at sub-lethal concentrations, showed a synergistic effect and resulted in a total inhibition of cells growth after 5 days. The results illustrate the efficacy of nisin and potassium sorbate in combination, and the commercial potential of applying such treatment to decontaminate any product that has a problem with persistence of bacterial spores.

  7. Spores from mesophilic Bacillus cereus strains germinate better and grow faster in simulated gastro-intestinal conditions than spores from psychrotrophic strains.

    NARCIS (Netherlands)

    Wijnands, L M; Dufrenne, J B; Zwietering, M H; Leusden, F M van

    2006-01-01

    The species Bacillus cereus, known for its ability to cause food borne disease, consists of a large variety of strains. An important property for discrimination of strains is their growth temperature range. Psychrotrophic strains can grow well at refrigerator temperatures but grow at 37 degrees C wi

  8. Spores from mesophilic Bacillus cereus strains germinate better and grow faster in simulated gastro-intestinal conditions than spores from psychrotrophic strains

    NARCIS (Netherlands)

    Wijnands, L.M.; Dufrenne, J.B.; Zwietering, M.H.; Leusden, van F.M.

    2006-01-01

    The species Bacillus cereus, known for its ability to cause food borne disease, consists of a large variety of strains. An important property for discrimination of strains is their growth temperature range. Psychrotrophic strains can grow well at refrigerator temperatures but grow at 37 °C with diff

  9. Field evaluation of a bioregulator containing live Bacillus cereus spores on health status and performance of sows and their litters.

    Science.gov (United States)

    Alexopoulos, C; Karagiannidis, A; Kritas, S K; Boscos, C; Georgoulakis, I E; Kyriakis, S C

    2001-04-01

    The efficacy of Paciflor, a bioregulator containing live Bacillus cereus CIP 5832 spores, was assessed in sows during late pregnancy and lactation, as well as in their piglets up to the growing phase. Two groups each of 30 pregnant gilts and sows received normal feed (T1 group), or feed with 85 g Paciflor per ton feed (T2 group), from 15 days prior to farrowing up to the end of the lactation period. Furthermore, 15 litters of the T1 group and 15 litters of the T2 group, were offered normal feed from the 5th to the 70th days of life (T1.1 and T2.1 groups, respectively), while the remaining 15 litters each of the T1 and T2 groups received the same feed but including Paciflor at a dose of 100 g/ton (from day 5 to day 49) and 50 g/ton (from day 50 to day 70). These pig litters were T1.2 and T2.2, respectively. No differences were seen between the T1 and T2 groups with respect to the clinical observations (loss of appetite, fever, mastitis, metritis and returns to oestrus, treatments applied, deaths, or removals to the slaughterhouse), gestation length, bodyweight of sows at farrowing or litter-size at birth. However, during lactation, the fat content of the dam's milk was increased (0.46% more fat), the body weight loss of sows was reduced and the number of weaned pigs per sow was increased (0.6 more pigs per litter) after administration of Paciflor (P pigs originating from Paciflor-treated dams (T2.2 group) (P < 0.05). Despite the fact that no difference was seen between groups with regard to the amount of feed consumed, the feed conversion ratio of Paciflor-treated piglets (T2.2 and T1.2) was significantly improved compared to that of the untreated piglets (T2.1 and T1.1) (P < 0.05). With respect to weight gain, for the Paciflor-treated piglets, those born to Paciflor-treated mothers (T2.2) were 0.56 kg heavier than those born to untreated dams (T1.2) (P < 0.05). It is concluded that administration of Paciflor in dams during the end of pregnancy and during lactation

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

    Science.gov (United States)

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

    2016-01-06

    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 10-10(4)-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.

  11. Cooperative manganese (II) activation of 3-phosphoglycerate mutase of Bacillus megaterium: a biological pH-sensing mechanism in bacterial spore formation and germination.

    Science.gov (United States)

    Kuhn, N J; Setlow, B; Setlow, P; Cammack, R; Williams, R

    1995-06-20

    The conversion of 3-P-glycerate mutase of Bacillus megaterium from a catalytically inactive to an active form was markedly more effective with buffered Mn2+ than with just added Mn2+. The previously reported stimulation by threonine disappeared when buffered Mn2+ was used. Activation of mutase showed a sigmoid dependence on Mn2+ concentration when buffered with tetramethylenediamine tetraacetate. The curve obeyed Hill kinetics with a coefficient of 2.1 +/- 0.1. At 0.5 microM free Mn2+, buffered with trimethylenediamine tetraacetate, activation of mutase increased about 73-fold over the pH range 6.6 to 7.4. Plotted against [OH-], the activation showed a strongly sigmoid response with Hill coefficient of 3.5 +/- 0.1. When mutase activated at pH 6.4 and 0.5 microM free Mn2+ in the presence of substrate was transferred to a similar medium at pH 7.4, the rate of product accumulation increased 360-fold within a few minutes. The pH sensitivity conferred upon mutase by low [Mn2+] may account for its large activity decrease during sporulation, and later increase during spore germination, when spore pH, respectively, declines and rises by about 1 unit. These changes result in the accumulation, and later reutilization, of 3-P-glycerate reserves in the spore. Such a pH-sensing function of Mn2+ may have wider biological uses.

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

    Science.gov (United States)

    2012-11-01

    defective in these spores. The original gerP mutation was in the gerPC gene, the third gene in the likely hexacistronic gerP operon ; a similar operon is...present in other Bacillales species. Mutations in individual genes of the B. subtilis gerP operon also reduce spore germination with nutrient germinants...but do not reduce spore viability, and deletion of the whole operon gives the same general phenotype as do mutations in individual gerP genes. The B

  13. Spore UV and acceleration resistance of endolithic Bacillus pumilus and Bacillus subtilis isolates obtained from Sonoran desert basalt: implications for lithopanspermia.

    Science.gov (United States)

    Benardini, James N; Sawyer, John; Venkateswaran, Kasthuri; Nicholson, Wayne L

    2003-01-01

    Bacterial spores have been used as model systems for studying the theory of interplanetary transport of life by natural processes such as asteroidal or cometary impacts (i.e., lithopanspermia). Because current spallation theory predicts that near-surface rocks are ideal candidates for planetary ejection and surface basalts are widely distributed throughout the rocky planets, we isolated spore-forming bacteria from the interior of near-subsurface basalt rocks collected in the Sonoran desert near Tucson, Arizona. Spores were found to inhabit basalt at very low concentrations (basalt samples. Populations of purified spores prepared from the isolated strains were subjected to 254-nm UV and ballistics tests in order to assess their resistance to UV radiation and to extreme acceleration shock, two proposed lethal factors for spores during interplanetary transfer. Specific natural isolates of B. pumilus were found to be substantially more resistant to UV and extreme acceleration than were reference laboratory strains of B. subtilis, the benchmark organism, suggesting that spores of environmental B. pumilus isolates may be more likely to survive the rigors of interplanetary transfer.

  14. Survival of foodborne pathogenic bacteria (Bacillus cereus, Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, and Listeria monocytogenes) and Bacillus cereus spores in fermented alcoholic beverages (beer and refined rice wine).

    Science.gov (United States)

    Kim, S A; Kim, N H; Lee, S H; Hwang, I G; Rhee, M S

    2014-03-01

    Only limited information is available on the microbiological safety of fermented alcoholic beverages because it is still a common belief that such beverages do not provide a favorable environment for bacterial growth and survival. Thus, in this study, we examined the survival of major foodborne pathogens and spores in fermented alcoholic beverages. Foodborne pathogens (Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus) and B. cereus spores (initial population, 3 to 4 log CFU/ml) were inoculated separately into three types of beer and refined rice wine, which were then stored at 5 and 22°C. Bacterial counts were assayed periodically for up to 28 days. Vegetative B. cereus counts decreased rapidly, whereas B. cereus spore counts remained constant (P > 0.05) for a long period of time in all beverages. Vegetative B. cereus cells formed spores in beer at 5 and 22°C, and the spores survived for long periods. Among vegetative cells, E. coli O157:H7 had the highest survival (only 1.49 to 1.56 log reduction during 28 days in beer at 5°C). Beer and refined rice wine supported microbial survival from several days to several weeks. Our results appear to contradict the common belief that pathogens cannot survive in alcoholic beverages. Long-term survival of pathogens (especially B. cereus and E. coli O157:H7) in beer and refined rice wine should be taken into consideration by the manufacturers of these beverages. This study provides basic information that should help further research into microbial survival in alcoholic beverages and increase the microbiological safety regulation of fermented alcoholic beverages.

  15. Evaluation of the Efficacy of Methyl Bromide in the Decontamination of Building and Interior Materials Contaminated with Bacillus anthracis Spores

    Data.gov (United States)

    U.S. Environmental Protection Agency — Spreadsheets containing data for recovery of spores from different materials. Data on the fumigation parameters are also included. This dataset is associated with...

  16. 枯草芽胞杆菌芽胞表面展示外源蛋白的研究进展%Research Progress on Bacillus subtilis Spore Display of Recombinant Proteins

    Institute of Scientific and Technical Information of China (English)

    余小霞; 田健; 伍宁丰

    2013-01-01

    Bacillus subtilis is Gram-positive bacteria with biological safy. It can form spores with strong stress resistance in poor nutrient environment. The spore of Bacillus subtilis consists of 3 parts including the core, cortex and spore coat protein. Recently, the Bacillus subtilis spore coat proteins, such as CotB, CotC, CotG, CotX and OxdD, have been successfully used as vectors to display the antigen proteins, enzymes or reporter protein on the spore surfaces. The recombinant proteins on Bacillus subtilis spores usually have many advantages, such as good stability, easy purification and safety. Therefore, they can be used in medicine, food and feed industry, and other fields. It has a great application prospect. This review introduced in detail the molecular characteristics of Bacillus subtilis spores, the construction process of the expressive system on spore surface and its application prospect. Thus, the paper provided a foundation for the basic and applied research about spore surface display system.%枯草芽胞杆菌是一种生物安全的革兰氏阳性细菌,在营养匮乏的环境下,可形成具有强抗逆性的芽胞。枯草芽胞杆菌的芽胞由核心、皮层、孢子外套蛋白三部分组成,目前已成功利用枯草芽胞杆菌芽胞外套蛋白CotB、CotC、CotG、CotX和OxdD为载体,将酶蛋白、抗原蛋白或荧光标记蛋白等展示于芽胞表面。芽胞表面展示的蛋白通常具有较好的稳定性、易于纯化和安全性好等优点,可应用于医药、食品及饲料工业等领域,具有较大的应用前景。详细介绍了枯草芽胞杆菌芽胞的分子特点及芽胞表面展示系统的构建过程及其应用前景,为芽胞表面展示载体的基础及应用研究奠定基础。

  17. Inactivation of Bacillus atrophaeus spores in healthcare waste by uv light coupled with H2O2

    Directory of Open Access Journals (Sweden)

    M. T. Iannotti

    2013-09-01

    Full Text Available Healthcare waste inoculated with B. atropheaus spores was used to evaluate a treatment process using UV light in combination with H2O2. First, the influence of the waste mass on the spore inactivation fraction was investigated for a constant radiation exposure time of 10 min and power per unit mass of waste (44-237 W/kg. The degree of inactivation of the spores was then determined as a function of exposure time (5-30 min and power per mass unit (67-178 W/kg for a constant waste mass. The experimental results were adjusted according to four kinetic models. The Hom and power law models were the most appropriate for the description of the disinfection process. The maximum experimental inactivation fraction (95% achieved was obtained with 178 W/kg irradiation for 30 min.

  18. Germination of Bacillus anthracis spores:research advances%炭疽芽孢杆菌芽孢萌发研究进展

    Institute of Scientific and Technical Information of China (English)

    高志奇; 刘先凯; 王恒樑

    2014-01-01

    芽孢是炭疽芽孢杆菌为应对不适的外界环境而形成的一种生命形式,休眠期的芽孢可以通过萌发恢复生长成为繁殖体。萌发过程作为关键步骤,可以由营养萌发剂和一些非营养类物质或者在其他情况下触发。在萌发过程中,萌发剂通过与存在于芽孢内膜上的萌发剂受体结合来触发芽孢核内各种阳离子的释放以及芽孢核对水的吸收。在芽孢皮层的肽聚糖被酶水解后,芽孢核逐渐完全水合化,开始进行新陈代谢以及大分子的合成活动,逐渐成长为一个新的营养细胞。该文将从萌发受体、芽孢皮层水解酶功能等方面对炭疽菌芽孢萌发机制进行阐述。%A spore is another life cycle form of Bacillus anthracis for resisting starvation.When conditions are favorable for growth, the dormant spore will germinate,go through outgrowth, and are ultimately converted back into a growing cell. As the first step back to vegetative growth, germination could be induced by nutrients and a variety of non-nutrient agents. Nutrient germinants trigger cation release and water absorption by binding to receptors in the spore′s inner membrane.Then the spore′s peptidoglycan cortex is hydrolyzed and the spore core rehydrates, which allows the resumption of spore metabo-lism and macromolecular synthesis.This paper reviews the nutrient germinant receptor and cortex lytic enzymes in the spore germination process of B.anthracis.

  19. The mechanism of DNA ejection in the Bacillus anthracis spore-binding phage 8a revealed by cryo-electron tomography

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Xiaofeng [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States); Walter, Michael H. [Department of Biology, University of Northern Iowa, Cedar Falls, IA 50614 (United States); Paredes, Angel [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States); Morais, Marc C., E-mail: mcmorais@utmb.edu [Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555 (United States); Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555 (United States); Liu, Jun, E-mail: Jun.Liu.1@uth.tmc.edu [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States)

    2011-12-20

    The structure of the Bacillus anthracis spore-binding phage 8a was determined by cryo-electron tomography. The phage capsid forms a T = 16 icosahedron attached to a contractile tail via a head-tail connector protein. The tail consists of a six-start helical sheath surrounding a central tail tube, and a structurally novel baseplate at the distal end of the tail that recognizes and attaches to host cells. The parameters of the icosahedral capsid lattice and the helical tail sheath suggest protein folds for the capsid and tail-sheath proteins, respectively, and indicate evolutionary relationships to other dsDNA viruses. Analysis of 2518 intact phage particles show four distinct conformations that likely correspond to four sequential states of the DNA ejection process during infection. Comparison of the four observed conformations suggests a mechanism for DNA ejection, including the molecular basis underlying coordination of tail sheath contraction and genome release from the capsid.

  20. Inactivation kinetics of Bacillus subtilis spores with ozone%臭氧灭活水中枯草芽孢杆菌的动力学

    Institute of Scientific and Technical Information of China (English)

    刘枫; 昌盛; 陈忠林

    2016-01-01

    以枯草芽孢杆菌(ATCC6633)的孢子作为难灭活微生物的代表,研究了消毒剂浓度和反应时间的乘积值(CT值)、pH值、温度对臭氧灭活水中芽孢效果的影响,并探讨了相关灭活反应的动力学特征.结果表明,臭氧灭活芽孢的过程可分为延滞期和灭活期,其灭活反应符合Chick-Watson延迟反应动力学模型.在半连续流反应模式下,当臭氧浓度在0.42-4.00 mg· L-1,反应时间0-20 min,pH值6-8,温度1-30℃范围内时,臭氧对芽孢的灭活效果与臭氧的CT值显著相关,与单独的臭氧浓度无关,CT值越高,所能达到的灭活率也越高.同时,温度对反应速率常数k影响较大,即随着温度的升高,灭活反应的延滞期CT1ag显著减小,反应速率常数k增大,臭氧对芽孢的灭活能力增强;而反应速率常数k在各pH值下基本不变,pH值对芽孢的灭活影响甚微.%In general,spores of Bacillus subtilis (ATCC6633) would be used as potential model for the resistant microorganisms.In this study,the inactivation kinetics of spores in drinking water by ozone was investigated,and the factors such as ozone such as the numerical value of the product of the concentration of ozone and the reaction time (CT) values,pH,and temperature which could influence the inactivation process were evaluated.Results showed that the inactivation process of spores with ozone was characterized by a lag phase followed by a logarithmic inactivation phase,and the delayed Chick-Watson model could well describe the inactivation process.In this study,the disinfection of Bacillus subtilis spores was performed in a semi-batch reactor under the conditions with the ozone concentration,reaction time,pH,and temperature ranged in 0.42-4.00 mg·L-1,0-20 min,6-8,and 1-30 ℃,respectively.It showed that the inactivation is independent of ozone dose and is obvious relative to the ozone CT values.A higher inactivation level of spores wou ld be achieved at higher CT values.In addition

  1. Impact of sorbic acid and other mild preservation stresses on germination and outgrowth of Bacillus cereus spores

    NARCIS (Netherlands)

    Melis, van C.C.J.

    2013-01-01

      Weak organic acids such as sorbic acid, lactate, and acetic acid are widely used by the food industry as preservatives to control growth of micro-organisms. With the current trend towards milder processing of food products, opportunities arise for spore-forming spoilage and pathogenic microo

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

    Science.gov (United States)

    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...

  3. Characterization of Bacillus sporothermodurans IC4 spores; putative indicator microorganism for optimisation of thermal processes in food sterilisation

    NARCIS (Netherlands)

    van Zuijlen, A.; Periago, P.M.; Amézquita, A.; Palop, A.; Brul, S.; Fernández, P.S.

    2010-01-01

    Spore-forming bacteria with high heat resistance increasingly challenge industrial sterilisation processes in foods. To ensure stability of manufactured foods, generally worst case scenarios are applied often leading to unwanted over processing of foods. This means bigger requirements of energy and

  4. Fate of Bacillus thuringiensis subsp. israelensis in the field: evidence for spore recycling and differential persistence of toxins in leaf litter.

    Science.gov (United States)

    Tetreau, Guillaume; Alessi, Mattia; Veyrenc, Sylvie; Périgon, Sophie; David, Jean-Philippe; Reynaud, Stéphane; Després, Laurence

    2012-12-01

    Bacillus thuringiensis subsp. israelensis is a bioinsecticide increasingly used worldwide for mosquito control. Despite its apparent low level of persistence in the field due to the rapid loss of its insecticidal activity, an increasing number of studies suggested that the recycling of B. thuringiensis subsp. israelensis can occur under specific, unknown conditions. Decaying leaf litters sampled in mosquito breeding sites in the French Rhône-Alpes region several months after a treatment were shown to exhibit a high level of larval toxicity and contained large amounts of spores. In the present article, we show that the high concentration of toxins found in these litters is consistent with spore recycling in the field, which gave rise to the production of new crystal toxins. Furthermore, in these toxic leaf litter samples, Cry4Aa and Cry4Ba toxins became the major toxins instead of Cyt1Aa in the commercial mixture. In a microcosm experiment performed in the laboratory, we also demonstrated that the toxins, when added in their crystal form to nontoxic leaf litter, exhibited patterns of differential persistence consistent with the proportions of toxins observed in the field-collected toxic leaf litter samples (Cry4 > Cry11 > Cyt). These results give strong evidence that B. thuringiensis subsp. israelensis recycled in specific breeding sites containing leaf litters, and one would be justified in asking whether mosquitoes can become resistant when exposed to field-persistent B. thuringiensis subsp. israelensis for several generations.

  5. Observations on the Inactivation Efficacy of a MALDI-TOF MS Chemical Extraction Method on Bacillus anthracis Vegetative Cells and Spores.

    Directory of Open Access Journals (Sweden)

    Simon A Weller

    Full Text Available A chemical (ethanol; formic acid; acetonitrile protein extraction method for the preparation of bacterial samples for matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS identification was evaluated for its ability to inactivate bacterial species. Initial viability tests (with and without double filtration of the extract through 0.2 μM filters, indicated that the method could inactivate Escherichia coli MRE 162 and Klebsiella pneumoniae ATCC 35657, with or without filtration, but that filtration was required to exclude viable, avirulent, Bacillus anthracis UM23CL2 from extracts. Multiple, high stringency, viability experiments were then carried out on entire filtered extracts prepared from virulent B. anthracis Vollum vegetative cells and spores ranging in concentration from 10(6-10(8 cfu per extract. B. anthracis was recovered in 3/18 vegetative cell extracts and 10/18 spore extracts. From vegetative cell extracts B. anthracis was only recovered from extracts that had undergone prolonged Luria (L-broth (7 day and L-agar plate (a further 7 days incubations. We hypothesise that the recovery of B. anthracis in vegetative cell extracts is due to the escape of individual sub-lethally injured cells. We discuss our results in view of working practises in clinical laboratories and in the context of recent inadvertent releases of viable B. anthracis.

  6. Observations on the Inactivation Efficacy of a MALDI-TOF MS Chemical Extraction Method on Bacillus anthracis Vegetative Cells and Spores.

    Science.gov (United States)

    Weller, Simon A; Stokes, Margaret G M; Lukaszewski, Roman A

    2015-01-01

    A chemical (ethanol; formic acid; acetonitrile) protein extraction method for the preparation of bacterial samples for matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) identification was evaluated for its ability to inactivate bacterial species. Initial viability tests (with and without double filtration of the extract through 0.2 μM filters), indicated that the method could inactivate Escherichia coli MRE 162 and Klebsiella pneumoniae ATCC 35657, with or without filtration, but that filtration was required to exclude viable, avirulent, Bacillus anthracis UM23CL2 from extracts. Multiple, high stringency, viability experiments were then carried out on entire filtered extracts prepared from virulent B. anthracis Vollum vegetative cells and spores ranging in concentration from 10(6)-10(8) cfu per extract. B. anthracis was recovered in 3/18 vegetative cell extracts and 10/18 spore extracts. From vegetative cell extracts B. anthracis was only recovered from extracts that had undergone prolonged Luria (L)-broth (7 day) and L-agar plate (a further 7 days) incubations. We hypothesise that the recovery of B. anthracis in vegetative cell extracts is due to the escape of individual sub-lethally injured cells. We discuss our results in view of working practises in clinical laboratories and in the context of recent inadvertent releases of viable B. anthracis.

  7. Lipid metabolism during bacterial growth, sporulation, and germination: differential synthesis of individual branched- and normal-chain fatty acids during spore germination and outgrowth of Bacillus thuringiensis.

    Science.gov (United States)

    Nickerson, K W; Bulla, L A; Mounts, T L

    1975-12-01

    The biosynthesis of individual branched- and normal-chain fatty acids during Bacillus thuringiensis spore germination and outgrowth was studied by comparing pulsed and continuous labeling of these fatty acids with [U-14C]acetate. The relative specific activity of each fatty acid varies with time as the cell progresses through outgrowth. However, fatty acid synthesis does occur in two distinct phases. Upon germination, acetate is incorporated only into the iso-isomers i-C13, i-C14, and i-C16; no normal or anteiso synthesis occurs. Subsequent to T30, the full complement of branched- and normal-chain homologues is formed and there is a dramatic enhancement in the overall rate of fatty acid synthesis. Significantly, this rate increase coincides with a marked shift from the synthesis of short-chain to long-chain fatty acids. These findings illustrate a dichotomy in synthesis that may result from initial fatty acid formation by preexisting spore fatty acid biosynthetic enzymes in the absence of de novo protein synthesis. Elucidation of the timing and kinetics of individual fatty acid formation provides a biochemical profile of activities directly related to membrane differentiation and cellular development.

  8. Lipid metabolism during bacterial growth, sporulation, and germination: kinetics of fatty acid and macromolecular synthesis during spore germination and outgrowth of Bacillus thuringiensis.

    Science.gov (United States)

    Nickerson, K W; De Pinto, J; Bulla, L A

    1975-01-01

    The timing and kinetics of fatty acid synthesis are delineated for Bacillus thuringiensis spore germination and outgrowth by analyzing [U-14C]acetate and [2-3H]glycerol incorporation into chloroform-methanol-extractable and trichloroacetic acid-precipitable lipids. In addition to measurement of pulsed and continuous labeling of fatty acids, monitoring the incorporation of radioactive phenylalanine, thymidine, and uridine from the onset of germination through first cell division provides a profile of biochemical activities related to membrane differentiation and cellular development. Upon germination, ribonucleic acid synthesis is initiated, immediately followed by rapid and extensive fatty acid synthesis that in turn precedes protein, deoxyribonucleic acid and triglyceride synthesis. Significantly, formation of fatty acids from acetate exhibits further developmental periodicity in which a large transient increase in fatty acid synthetic activity coincides with the approach of cell division. Radiorespirometric analyses indicates only slight oxidative decarboxylation of acetate and corroborates the extreme involvement of acetate in specific fatty acid biosynthetic reactions throughout cellular modification. These findings graphically demonstrate an intimate association of fatty acid metabolism with commitment to spore outgrowth and subsequent cell division.

  9. Inactivation kinetics of spores of Bacillus cereus strains treated by a peracetic acid-based disinfectant at different concentrations and temperatures.

    Science.gov (United States)

    Sudhaus, Nadine; Pina-Pérez, Maria Consuelo; Martínez, Antonio; Klein, Günter

    2012-05-01

    The purpose of this study was to assess the effect of a commercial peracetic acid-based disinfectant against spores of Bacillus cereus, to identify the most influential factor for the final number of microorganisms after different disinfection procedures, and to evaluate the nature of the inactivation kinetics. The spores of four different strains of B. cereus (DSM 318, 4312, 4313, and 4384) were treated with five different disinfectant concentrations (0.25%, 0.5%, 1.0%, 1.5%, and 2.0% [w/v]) at three different temperatures (10°C, 15°C, and 20°C) with or without protein load. A higher temperature and PES 15/23 concentration resulted in a higher inactivation. Inactivation of B. cereus strain 4312 was around 2 log₁₀ cycles at 10°C and around 7 log₁₀ at 20°C (conc=1% [w/v] PAA; t=60 min; without protein). The protein load at higher concentrations did not significantly reduce the efficacy of the disinfectant (p>0.05). This article indicates the applicability of the Weibull model to fit the B. cereus disinfectant survival curves. A Monte Carlo simulation was used to carry out a sensitivity analysis, which revealed the most influential factors affecting the final number of microorganisms after the disinfection process.

  10. Decontamination efficacy of three commercial-off-the-shelf (COTS sporicidal disinfectants on medium-sized panels contaminated with surrogate spores of Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Jason M Edmonds

    Full Text Available In the event of a wide area release and contamination of a biological agent in an outdoor environment and to building exteriors, decontamination is likely to consume the Nation's remediation capacity, requiring years to cleanup, and leading to incalculable economic losses. This is in part due to scant body of efficacy data on surface areas larger than those studied in a typical laboratory (5×10-cm, resulting in low confidence for operational considerations in sampling and quantitative measurements of prospective technologies recruited in effective cleanup and restoration response. In addition to well-documented fumigation-based cleanup efforts, agencies responsible for mitigation of contaminated sites are exploring alternative methods for decontamination including combinations of disposal of contaminated items, source reduction by vacuuming, mechanical scrubbing, and low-technology alternatives such as pH-adjusted bleach pressure wash. If proven effective, a pressure wash-based removal of Bacillus anthracis spores from building surfaces with readily available equipment will significantly increase the readiness of Federal agencies to meet the daunting challenge of restoration and cleanup effort following a wide-area biological release. In this inter-agency study, the efficacy of commercial-of-the-shelf sporicidal disinfectants applied using backpack sprayers was evaluated in decontamination of spores on the surfaces of medium-sized (∼1.2 m2 panels of steel, pressure-treated (PT lumber, and brick veneer. Of the three disinfectants, pH-amended bleach, Peridox, and CASCAD evaluated; CASCAD was found to be the most effective in decontamination of spores from all three panel surface types.

  11. Array lead zirconate titanate/glass piezoelectric microcantilevers for real-time detection of Bacillus anthracis with 10 spores/ml sensitivity and 1/1000 selectivity in bacterial mixtures

    Science.gov (United States)

    McGovern, John-Paul; Shih, Wei-Heng; Rest, Richard F.; Purohit, Mitali; Mattiucci, Mark; Pourrezaei, Kambiz; Onaral, Banu; Shih, Wan Y.

    2009-12-01

    An array of three identical piezoelectric microcantilever sensors (PEMSs) consisting of a lead zirconate titanate layer bonded to a glass layer was fabricated and examined for simultaneous, in situ, real-time, all-electrical detection of Bacillus anthracis (BA) spores in an aqueous suspension using the first longitudinal extension mode of resonance. With anti-BA antibody immobilized on the sensor surfaces all three PEMS exhibited identical BA detection resonance frequency shifts at all tested concentrations, 10-107 spores/ml with a standard deviation of less than 10%. The detection concentration limit of 10 spores/ml was about two orders of magnitude lower than would be permitted by flexural peaks. In blinded-sample testing, the array PEMS detected BA in three samples containing BA: (1) 3.3×103 spores/ml, (2) a mixture of 3.3×103 spores/ml and 3.3×105 S. aureus (SA) and P. aeruginosa (PA) per ml, and (3) a mixture of 3.3×103 spores/ml with 3.3×106 SA+PA/ml. There was no response to a sample containing only 3.3×106 SA+PA/ml. These results illustrate the sensitivity, specificity, reusability, and reliability of array PEMS for in situ, real-time detection of BA spores.

  12. Optimization of spore producing fermentation conditions of probiotic strain bacillus velezensis Z-27 from pig%猪源益生菌株Bacillus velezensisZ-27产芽孢发酵条件优化

    Institute of Scientific and Technical Information of China (English)

    王振海; 姜军坡; 王选; 王世英; 朱宝成

    2011-01-01

    Spore producing fermentation conditions of probiotic strain bacillus velezensis Z-27 from pig were optimized. With spore yield and biomass as main indicators, single factor test and orthogonal test were done to determine the suitable conditions for fermentation in shake flask. Results showed that biomass was 2.33 x1010 cfu/ml and spore yield was 96.0% at the optimal condition that corn meal was 1%, soybean meal 1.5%, MnSO4 0.03%,, NaH2PO4·2H2O 0.2%, Na2HPO4·2H2O 0.4%, initial pH 7.0, media volume 50 ml/250 ml, inoculation volume 2%, rotation speed 220 r/min, cultured for 24 h at 37 °C. It laid foundation for further research and application of strain bacillus velezensis Z-27.%对猪源益生菌株Bacillus velezensis Z-27进行发酵条件优化试验.以芽孢产率和生物量为指标,利用单因素试验和正交试验确定了摇瓶发酵的最佳条件:玉米粉1%、豆饼粉1.5%、MnSO40.03%、NaH2PO4·2H2O 0.2%、Na2HPO4·2H2O 0.4%,初始pH值7.0,250 ml三角瓶装量50 ml,接种量2%,摇床转速220 r/min,发酵温度37℃.优化后发酵液的生物量为2.33×1010cfu/ml,芽孢产率达96.0%,为该菌株的进一步研究和应用奠定了基础.

  13. The effects of various cure cycles upon the viability of Bacillus subtillis var. niger spores within solid propellant.

    Science.gov (United States)

    Brewer, W. A.; Paik, W. W.; Robillard, C. L.; Green, R. H.; Smith, C. D.

    1972-01-01

    Saturethane solid propellant was used in all tests. The spore inoculum was evenly distributed in the propellant. Samples weighing approximately 5 g were aseptically removed, placed into curing ovens, and exposed to cure temperatures. Initial tests were conducted at 82 and 93 C. Analysis of the obtained data indicated that the survivor curves were not linear. Exposure of the inoculated propellant samples to 93 C reduced the initial population to less than 0.01% in about 20 hours. At 82 C, approximately 168 hours were required for a similar reduction. Tests involving curing temperatures of 105 and 115 C were also conducted. It is pointed out that changes in the mechanism of spore inactivation due to chemical and physical changes in the propellant could account for the nonlinear survivor curves obtained.

  14. Roles of Macrophages and Neutrophils in the Early Host Response to Bacillus anthracis Spores in a Mouse Model of Infection

    Science.gov (United States)

    2006-01-01

    basophil and lymphocyte populations. In some experiments, control mice were pretreated with saline, and in others, rat IgG (reagent grade, from serum... International . REFERENCES 1. Abalakin, V. A., E. G. Sirina, and T. D. Cherkasova. 1990. The effect of lethal anthrax toxin on the functional activity of...The structure of spores as revealed by mechanical disruption. J. Bacteriol. 66:312–319. 16. Friedlander, A. M. 2000. Anthrax: clinical features

  15. Inactivation of vegetative cells, but not spores, of Bacillus anthracis, B. cereus, and B. subtilis on stainless steel surfaces coated with an antimicrobial silver- and zinc-containing zeolite formulation.

    Science.gov (United States)

    Galeano, Belinda; Korff, Emily; Nicholson, Wayne L

    2003-07-01

    Stainless steel surfaces coated with paints containing a silver- and zinc-containing zeolite (AgION antimicrobial) were assayed in comparison to uncoated stainless steel for antimicrobial activity against vegetative cells and spores of three Bacillus species, namely, B. anthracis Sterne, B. cereus T, and B. subtilis 168. Under the test conditions (25 degrees C and 80% relative humidity), the zeolite coating produced approximately 3 log(10) inactivation of vegetative cells within a 5- to 24-h period, but viability of spores of the three species was not significantly affected.

  16. False-negative rate, limit of detection and recovery efficiency performance of a validated macrofoam-swab sampling method for low surface concentrations of Bacillus anthracis Sterne and Bacillus atrophaeus spores

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, G. F. [Applied Statistics and Computational Sciences, Pacific Northwest National Laboratory, Richland WA USA; Deatherage Kaiser, B. L. [Chemical and Biological Signature Science Group, Pacific Northwest National Laboratory, Richland WA USA; Amidan, B. G. [Applied Statistics and Computational Sciences, Pacific Northwest National Laboratory, Richland WA USA; Sydor, M. A. [Chemical and Biological Signature Science Group, Pacific Northwest National Laboratory, Richland WA USA; Barrett, C. A. [Analytical Chemistry of Nuclear Materials, Pacific Northwest National Laboratory, Richland WA USA; Hutchison, J. R. [Chemical and Biological Signature Science Group, Pacific Northwest National Laboratory, Richland WA USA

    2016-05-06

    The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target amounts (2-500 spores) to positive-control plates and test coupons (2 in × 2 in) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest for vinyl tile (50.8% with BAS and 40.2% with BG) and the highest for glass (92.8% with BAS and 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG; values increased as concentration decreased in the range tested (0.078 to 19.375 CFU/cm2). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results will be discussed in a subsequent article.

  17. Inactivation of Escherichia coli, Bacteriophage MS2, and Bacillus Spores under UV/H2O2 and UV/Peroxydisulfate Advanced Disinfection Conditions.

    Science.gov (United States)

    Sun, Peizhe; Tyree, Corey; Huang, Ching-Hua

    2016-04-19

    Ultraviolet light (UV) combined with peroxy chemicals, such as H2O2 and peroxydisulfate (PDS), have been considered potentially highly effective disinfection processes. This study investigated the inactivation of Escherichia coli, bacteriophage MS2, and Bacillus subtilis spores as surrogates for pathogens under UV/H2O2 and UV/PDS conditions, with the aim to provide further understanding of UV-based advanced disinfection processes (ADPs). Results showed that one additional log of inactivation of E. coli was achieved with 0.3 mM H2O2 or PDS at 5.2 × 10(-5) Einstein·L(-1) photo fluence (at 254 nm) compared with UV irradiation alone. Addition of H2O2 and PDS greatly enhanced the inactivation rate of MS2 by around 15 folds and 3 folds, respectively, whereas the inactivation of B. subtilis spores was slightly enhanced. Reactive species responsible for the inactivation were identified to be •OH, SO4(·-), and CO3(·-) based on manipulation of solution conditions. The CT value of each reactive species was calculated with respect to each microbial surrogate, which showed that the disinfection efficacy ranked as •OH > SO4(·-) > CO3(·-) ≫ O2(·-)/HO2(·). A comprehensive dynamic model was developed and successfully predicted the inactivation of the microbial surrogates in surface water and wastewater matrices. The concepts of UV-efficiency and EE/O were employed to provide a cost-effective evaluation for UV-based ADPs. Overall, the present study suggests that it will be beneficial to upgrade UV disinfection to UV/H2O2 ADP for the inactivation of viral pathogens.

  18. Fluorescence spectra and biological activity of aerosolized bacillus spores and MS2 bacteriophage exposed to ozone at different relative humidities in a rotating drum

    Science.gov (United States)

    Ratnesar-Shumate, Shanna; Pan, Yong-Le; Hill, Steven C.; Kinahan, Sean; Corson, Elizabeth; Eshbaugh, Jonathan; Santarpia, Joshua L.

    2015-03-01

    Biological aerosols (bioaerosols) released into the environment may undergo physical and chemical transformations when exposed to atmospheric constituents such as solar irradiation, reactive oxygenated species, ozone, free radicals, water vapor and pollutants. Aging experiments were performed in a rotating drum chamber subjecting bioaerosols, Bacillus thuringiensis Al Hakam (BtAH) spores and MS2 bacteriophages to ozone at 0 and 150 ppb, and relative humidities (RH) at 10%, 50%, and 80+%. Fluorescence spectra and intensities of the aerosols as a function of time in the reaction chamber were measured with a single particle fluorescence spectrometer (SPFS) and an Ultra-Violet Aerodynamic Particle Sizer® Spectrometer (UV-APS). Losses in biological activity were measured by culture and quantitative polymerase chain reaction (q-PCR) assay. For both types of aerosols the largest change in fluorescence emission was between 280 and 400 nm when excited at 263 nm followed by fluorescence emission between 380 and 700 nm when excited at 351 nm. The fluorescence for both BtAH and MS2 were observed to decrease significantly at high ozone concentration and high RH when excited at 263 nm excitation. The decreases in 263 nm excited fluorescence are indicative of hydrolysis and oxidation of tryptophan in the aerosols. Fluorescence measured with the UV-APS (355-nm excitation) increased with time for both BtAH and MS2 aerosols. A two log loss of MS2 bacteriophage infectivity was observed in the presence of ozone at ~50% and 80% RH when measured by culture and normalized for physical losses by q-PCR. Viability of BtAH spores after exposure could not be measured due to the loss of genomic material during experiments, suggesting degradation of extracelluar DNA attributable to oxidation. The results of these studies indicate that the physical and biological properties of bioaerosols change significantly after exposure to ozone and water vapor.

  19. [Raman spectra and structure analysis of 2,6-pyridine dicarboxylic acid in different states and single Bacillus spore].

    Science.gov (United States)

    Huang, Rong-shao; Huang, Xi; Xu, Lan-lan; Li, Yong-qing; Huang, Shu-shi

    2011-03-01

    The Raman spectra of 2,6-pyridine dicarboxylic acid (DPA) and their calcium salts(Ca-DPA) in different states and the Ca-DPA in a single bacterial spore have been recorded by laser tweezers Raman system (LTRS) and the spectra have been assigned. Raman spectra of different states of DPA and Ca-DPA are different evidently. Analysis leading to differences in the structure of spectrum may be due to that the Raman spectra of DPA crystalline reflected more precise characteristics information compared to DPA powder, in which the laser can penetrate through DPA crystalline and the Raman scatter from the crystalline interior is greater than that from DPA powder. The second reason is that DPA powder and Ca-DPA crystalline contain water molecules, and the intermolecular hydrogen bonding in the crystals of these molecules is extensive. The presence of calcium ions would affect the pyridine ring so that both sides of the carboxyl pyridine ring have a certain geometric deformation and the hydroxy carboxylic was damaged. The DPA2-anion is principal in Ca-DPA and the DPA solution. The calcium ion affects the stability of the pyridine ring structure in the Ca-DPA solution. The result from the spectra also showed that the DPA in single spores present Ca-DPA crystal state.

  20. Investigating synergism during sequential inactivation of MS-2 phage and Bacillus subtilis spores with UV/H2O2 followed by free chlorine.

    Science.gov (United States)

    Cho, Min; Gandhi, Varun; Hwang, Tae-Mun; Lee, Sangho; Kim, Jae-Hong

    2011-01-01

    A sequential application of UV as a primary disinfectant with and without H(2)O(2) addition followed by free chlorine as secondary, residual disinfectant was performed to evaluate the synergistic inactivation of selected indicator microorganisms, MS-2 bacteriophage and Bacillus subtilis spores. No synergism was observed when the UV irradiation treatment was followed by free chlorine, i.e., the overall level of inactivation was the same as the sum of the inactivation levels achieved by each disinfection step. With the addition of H(2)O(2) in the primary UV disinfection step, however, enhanced microbial inactivation was observed. The synergism was observed in two folds manners: (1) additional inactivation achieved by hydroxyl radicals generated from the photolysis of H(2)O(2) in the primary UV disinfection step, and (2) damage to microorganisms in the primary step which facilitated the subsequent chlorine inactivation. Addition of H(2)O(2) in the primary disinfection step was also found to be beneficial for the degradation of selected model organic pollutants including bisphenol-A (endocrine disruptor), geosmin (taste and odor causing compound) and 2,4-D (herbicide). The results suggest that the efficiency of UV/free chlorine sequential disinfection processes, which are widely employed in drinking water treatment, could be significantly enhanced by adding H(2)O(2) in the primary step and hence converting the UV process to an advanced oxidation process.

  1. Evaluation of the Relationship between the Adenosine Triphosphate (ATP Bioluminescence Assay and the Presence of Bacillus anthracis Spores and Vegetative Cells

    Directory of Open Access Journals (Sweden)

    Shawn G. Gibbs

    2014-05-01

    Full Text Available Background: The Adenosine triphosphate (ATP bioluminescence assay was utilized in laboratory evaluations to determine the presence and concentration of vegetative and spore forms of Bacillus anthracis Sterne 34F2. Methods: Seventeen surfaces from the healthcare environment were selected for evaluation. Surfaces were inoculated with 50 µL of organism suspensions at three concentrations of 104, 106, 108 colony forming units per surface (CFU/surface of B. anthracis. Culture-based methods and ATP based methods were utilized to determine concentrations. Results: When all concentrations were evaluated together, a positive correlation between log-adjusted CFU and Relative Light Units (RLU for endospores and vegetative cells was established. When concentrations were evaluated separately, a significant correlation was not demonstrated. Conclusions: This study demonstrated a positive correlation for ATP and culture-based methods for the vegetative cells of B. anthracis. When evaluating the endospores and combining both metabolic states, the ATP measurements and CFU recovered did not correspond to the initial concentrations on the evaluated surfaces. The results of our study show that the low ATP signal which does not correlate well to the CFU results would not make the ATP measuring devises effective in confirming contamination residual from a bioterrorist event.

  2. Evaluation of Cytotoxicity, Genotoxicity and Hematotoxicity of the Recombinant Spore-Crystal Complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis in Swiss Mice

    Science.gov (United States)

    de Souza Freire, Ingrid; Miranda-Vilela, Ana Luisa; Barbosa, Lilian Carla Pereira; Martins, Erica Soares; Monnerat, Rose Gomes; Grisolia, Cesar Koppe

    2014-01-01

    The insecticidal properties of Cry-endotoxins from Bacillus thuringiensis (Bt) have long been used as spore-crystals in commercial spray formulations for insect control. Recently, some Bt-endotoxin genes have been cloned in many different plants. Toxicological evaluations of three spore-crystal endotoxins, BtCry1Ia, BtCry10Aa and BtCry1Ba6 from B. thuringiensis, were carried out on mice to understand their adverse effects on hematological systems and on genetic material. These three spore-crystals have shown toxic activity to the boll weevil, which is one of the most aggressive pests of the cotton crop. Cry1Ia, Cry10Aa and Cry1Ba6 did not increase the micronucleus frequency in the peripheral erythrocytes of mice and did not cause changes in the frequency of polychromatic erythrocytes. However, some hematologic disburbances were observed, specifically related to Cry1Ia and Cry1Ba6, respectively, for the erythroid and lymphoid lineage. Thus, although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results showed that these Bt spore-crystals were not harmless to mice, indicating that each spore-crystal endotoxin presents a characteristic profile of toxicity and might be investigated individually. PMID:25268978

  3. Evaluation of cytotoxicity, genotoxicity and hematotoxicity of the recombinant spore-crystal complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis in Swiss mice.

    Science.gov (United States)

    de Souza Freire, Ingrid; Miranda-Vilela, Ana Luisa; Barbosa, Lilian Carla Pereira; Martins, Erica Soares; Monnerat, Rose Gomes; Grisolia, Cesar Koppe

    2014-09-29

    The insecticidal properties of Cry-endotoxins from Bacillus thuringiensis (Bt) have long been used as spore-crystals in commercial spray formulations for insect control. Recently, some Bt-endotoxin genes have been cloned in many different plants. Toxicological evaluations of three spore-crystal endotoxins, BtCry1Ia, BtCry10Aa and BtCry1Ba6 from B. thuringiensis, were carried out on mice to understand their adverse effects on hematological systems and on genetic material. These three spore-crystals have shown toxic activity to the boll weevil, which is one of the most aggressive pests of the cotton crop. Cry1Ia, Cry10Aa and Cry1Ba6 did not increase the micronucleus frequency in the peripheral erythrocytes of mice and did not cause changes in the frequency of polychromatic erythrocytes. However, some hematologic disburbances were observed, specifically related to Cry1Ia and Cry1Ba6, respectively, for the erythroid and lymphoid lineage. Thus, although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results showed that these Bt spore-crystals were not harmless to mice, indicating that each spore-crystal endotoxin presents a characteristic profile of toxicity and might be investigated individually.

  4. Evaluation of Cytotoxicity, Genotoxicity and Hematotoxicity of the Recombinant Spore-Crystal Complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis in Swiss Mice

    Directory of Open Access Journals (Sweden)

    Ingrid de Souza Freire

    2014-09-01

    Full Text Available The insecticidal properties of Cry-endotoxins from Bacillus thuringiensis (Bt have long been used as spore-crystals in commercial spray formulations for insect control. Recently, some Bt-endotoxin genes have been cloned in many different plants. Toxicological evaluations of three spore-crystal endotoxins, BtCry1Ia, BtCry10Aa and BtCry1Ba6 from B. thuringiensis, were carried out on mice to understand their adverse effects on hematological systems and on genetic material. These three spore-crystals have shown toxic activity to the boll weevil, which is one of the most aggressive pests of the cotton crop. Cry1Ia, Cry10Aa and Cry1Ba6 did not increase the micronucleus frequency in the peripheral erythrocytes of mice and did not cause changes in the frequency of polychromatic erythrocytes. However, some hematologic disburbances were observed, specifically related to Cry1Ia and Cry1Ba6, respectively, for the erythroid and lymphoid lineage. Thus, although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results showed that these Bt spore-crystals were not harmless to mice, indicating that each spore-crystal endotoxin presents a characteristic profile of toxicity and might be investigated individually.

  5. Effect of modified atmosphere and temperature abuse on the growth from spores and cereulide production of Bacillus weihenstephanensis in a cooked chilled meat sausage.

    Science.gov (United States)

    Thorsen, Line; Budde, Birgitte Bjørn; Koch, Anette Granly; Klingberg, Trine Danø

    2009-04-15

    The effect of modified atmosphere packaging (MAP) on the germination and growth of toxin producing psychrotolerant Bacillus spp is not well described. A model agar system mimicking a cooked meat product was used in initial experiments. Incubation at refrigeration temperature of 8 degrees C for 5 weeks of 26 Bacillus weihenstephanensis including two emetic toxin (cereulide) producing strains showed that B. weihenstephanensis is sensitive to MAP containing CO2. The sensitivity to 20% CO2 was dependent on strain and oxygen level, being increased when oxygen was excluded from the MAP. Growth from spores was observed at the earliest within 2 weeks when 20% CO2 was combined with 2% O2 and in 3 weeks when combined with "0"% O2 (the remaining atmosphere was made up from N2). Results were validated in a cooked meat sausage model for two non-emetic and one emetic B. weihenstephanensis strain. The packaging film oxygen transfer rates (OTR) were 1.3 and 40 ml/m(2)/24 h and the atmospheres were 2% O2/20% CO2 and "0"% O2/20% CO2. Oxygen availability had a large impact on the growth from spores in the MAP meat sausage, only the most oxygen restricted condition (OTR of 1.3 ml/m(2)/24 h and "0"% O2/20 % CO2 inhibited growth of the three strains during 4 weeks storage at 8 degrees C. Cereulide production was undetectable during storage at 8 degrees C irrespective of choice of the MAP (quantified by liquid chromatography mass spectrometry/mass spectrometry). MAP storage at 8 degrees C for 1 and 3 weeks followed by opening of packages and temperature abuse for 1.5 h daily at 20 degrees C during 1 week resulted in increased cell counts and variable cereulide production in the meat sausage. A pre-history at 8 degrees C for 1 week in MAP with OTR of 1.3 or 40 ml/m(2)/24 h and 2% O2 resulted in cereulide concentrations of 0.816-1.353 microg/g meat sausage, while a pre-history under the most oxygen restricted condition (OTR of 1.3 ml/m(2)/24 h, "0"% O2/20 % CO2 resulted in minimal

  6. Drosophila melanogaster Selection for Survival after Infection with Bacillus cereus Spores: Evolutionary Genetic and Phenotypic Investigations of Respiration and Movement

    Directory of Open Access Journals (Sweden)

    Junjie Ma

    2013-01-01

    Full Text Available Laboratory populations of D. melanogaster have been subjected to selection for survival after live spores of B. cereus were introduced as a pathogenic agent. The present study was designed to investigate correlated traits: respiration as a metabolic trait and movement as a behavioral trait. An underlying hypothesis was that the evolution of increased survival after B. cereus infection exerts a metabolic cost associated with elevated immunity and this would be detected by increased respiration rates. There was support for this hypothesis in the male response to selection, but not for selected-line females. Two phenotypic effects were also observed in the study. Females especially showed a marked increase in respiration after mating compared to the other assay stages regardless of whether respiration was measured per fly or adjusted by lean mass or dry weight. Given that mating stimulates egg production, it is feasible that elevated metabolism was needed to provision oocytes with yolk. Females also moved less than males, perhaps due to behaviors related to oviposition whereas elevated male activity might be due to behaviors associated with seeking females and courtship. Relatively low movement of females indicated that their elevated respiration after mating was not due to a change in locomotion.

  7. Optimization of spore production condition of concrete self-healing bacterium Bacillus cohnii DSM6307%混凝土修复功能菌Bacillus cohnii DSM6307芽孢发酵条件优化

    Institute of Scientific and Technical Information of China (English)

    柯金龙; 彭慧; 刘冰; 邓旭; 邢锋

    2015-01-01

    对嗜碱科式芽孢杆菌( alkaliphilic Bacillus cohnii, DSM6307)芽孢形成的影响因素进行了研究.单因素实验结果表明,蔗糖为最适碳源,最佳质量浓度为1.0 g/L;牛肉膏为最适氮源,最佳质量浓度为3.0 g/L; Mn2+最适质量浓度为3.2 mg/L; Mg2+最适质量浓度为0.12 g/L;最适温度为30℃;装液量为50 mL (250 mL锥形瓶).运用Plackett-Burman法研究了碳源、氮源、微量元素、温度和装液量5个因素对DSM6307产芽孢数的影响,结果表明,碳源、氮源和Mn2+是影响DSM6307芽孢产量的显著因子.运用中心组合设计实验对这3种显著因子进行优化后,应用响应面模型测出蔗糖、牛肉膏和Mn2+的最优质量浓度分别为1.30 g/L、3.29 g/L和11.48 mg/L,预测芽孢数为1.67×109 mL-1.在此优化条件下,实验得到的芽孢数为1.50×109 mL-1,与预测值接近.%This paper investigates the influential factors of the spore production of alkaliphilic Bacillus cohnii (DSM6307). Firstly, we employed a single factor optimization method and obtained the results that the optimum carbon source is sucrose with the suitable concentration of 1. 0 g/L; the optimum nitrogen source is a beef extract with a suitable concentration of 3. 0 g/L;the optimal concentration of Mn2+ is 3. 2 mg/L, and the optimal concentra-tion of Mg2+ is 0. 12 g/L;the suitable temperature is 30 ℃, and the loading volume is 50 mL in a 250 mL conical flask. Then the Plackett-Burman design of the experiment reveals that the carbon source, nitrogen source and Mn2+are the significant factors among the 5 factors of carbon source, nitrogen source, microelement level, temperature and the loading volume. Finally, we performed further optimization with central composite design and response sur-face analysis, and it is found that the spore production of DSM6307 is 1. 67 × 109 mL-1 with optimized concentrations of sucrose, beef extract and Mn2+ being 1. 30 g/L, 3. 29 g/L and 11. 48 mg

  8. Hematotoxicity and genotoxicity evaluations in Swiss mice intraperitoneally exposed to Bacillus thuringiensis (var kurstaki) spore crystals genetically modified to express individually Cry1Aa, Cry1Ab, Cry1Ac, or Cry2Aa.

    Science.gov (United States)

    Mezzomo, Bélin Poletto; Miranda-Vilela, Ana Luisa; Barbosa, Lilian Carla Pereira; Albernaz, Vanessa Lima; Grisolia, Cesar Koppe

    2016-08-01

    Bacillus thuringiensis (Bt) has been widely used in foliar sprays as part of integrated pest management strategies against insect pests of agricultural crops. Since the advent of genetically modified plants expressing Bt δ-endotoxins, the bioavailability of Cry proteins has increased, and therefore for biosafety reasons their adverse effects should be studied, mainly for nontarget organisms. We evaluated, in Swiss mice, the hematotoxicity and genotoxicity of the genetically modified strains of Bt spore crystals Cry1Aa, 1Ab, 1Ac, or 2Aa at 27 mg/kg, and Cry1Aa, 1Ab and 2Aa also at 136 and 270 mg/kg, administered with a single intraperitoneal injection 24 h before euthanasia. Controls received filtered water or cyclophosphamide. Blood samples collected by cardiac puncture were used to perform hemogram, and bone marrow was extracted for the micronucleus test. Bt spore crystals presented toxicity for lymphocytes when in higher doses, which varied according to the type of spore crystal studied, besides promoting cytotoxic and genotoxic effects for the erythroid lineage of bone marrow, mainly at highest doses. Although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results indicated that these Bt spore crystals were not harmless to mice. This suggests that a more specific approach should be taken to increase knowledge about their toxicological properties and to establish the toxicological risks to nontarget organisms. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 970-978, 2016.

  9. Survival of spores of the UV-resistant Bacillus subtilis strain MW01 after exposure to low-earth orbit and simulated martian conditions: data from the space experiment ADAPT on EXPOSE-E.

    Science.gov (United States)

    Wassmann, Marko; Moeller, Ralf; Rabbow, Elke; Panitz, Corinna; Horneck, Gerda; Reitz, Günther; Douki, Thierry; Cadet, Jean; Stan-Lotter, Helga; Cockell, Charles S; Rettberg, Petra

    2012-05-01

    In the space experiment "Molecular adaptation strategies of microorganisms to different space and planetary UV climate conditions" (ADAPT), bacterial endospores of the highly UV-resistant Bacillus subtilis strain MW01 were exposed to low-Earth orbit (LEO) and simulated martian surface conditions for 559 days on board the European Space Agency's exposure facility EXPOSE-E, mounted outside the International Space Station. The survival of B. subtilis MW01 spores from both assays (LEO and simulated martian conditions) was determined by a colony-formation assay after retrieval. It was clearly shown that solar extraterrestrial UV radiation (λ≥110 nm) as well as the martian UV spectrum (λ≥200 nm) was the most deleterious factor applied; in some samples only a few spore survivors were recovered from B. subtilis MW01 spores exposed in monolayers. However, if shielded from solar irradiation, about 8% of MW01 spores survived in LEO conditions, and 100% survived in simulated martian conditions, compared to the laboratory controls. The results demonstrate the effect of shielding against the high inactivation potential of extraterrestrial solar UV radiation, which limits the chances of survival of even the highly UV-resistant strain of B. subtilis MW01 in the harsh environments of outer space and the martian surface.

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

    Science.gov (United States)

    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).

  11. Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination

    OpenAIRE

    2015-01-01

    Bacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organism Bacillus subtilis. In B. subtilis, the initiation of germinant receptor-mediated spore germination is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II is activated by the DPA released...

  12. 3种细菌芽胞遗传同源性及抗力对比研究%Comparison of Genetic Homology and Resistance of Three Bacillus Spores

    Institute of Scientific and Technical Information of China (English)

    许斌; 程立庆; 黄国荣; 张林; 饶中林; 张廷惠; 熊鸿燕

    2009-01-01

    比较细菌芽胞的遗传同源性、结构和抗力差异,为炭疽芽胞的替代试验菌的可靠评价提供依据.采用资料检索、生物信息分析、显微镜观察和微生物学技术分析不同芽胞的遗传同源性、超微结构和抗力差异.炭疽芽胞与腊样芽胞的结构和大小相似,生物遗传同源性最近,对热力、UVC和有效氯的抗力相近.腊样芽胞对炭疽芽胞的代表性最好,可以倾向性选用其替代炭疽芽胞进行试验研究.%Genetic homology, structure, and resistance difference of bacillus spores were compared to provide founda-tion to evaluate the reliability of experimental substitute for Bacillus anthracis spore using data-search, bio-information analysis, microscopic observation, and microbiological technology to analyze the genetic homology, ultrastructure, and resistance difference of different spores. The results showed that the structure and the size of B. anthracis was similar to those of B. cereus, very close in genetic homology, close in the resistance against heat, UVC, and effective chlo-rine. Therefore, B. cereus had the best representativeness of B. anthracis, and could be used tendentiously and selec-tively to study their spores.

  13. 傅里叶变换红外光谱对枯草芽孢杆菌的光学特性研究%Optical Properties Research of Bacillus Subtilis Spores by Fourier Transform Infrared Spectroscopy

    Institute of Scientific and Technical Information of China (English)

    冯明春; 徐亮; 高闽光; 焦洋; 魏秀丽; 金岭; 程巳阳; 李相贤; 冯书香

    2012-01-01

    使用傅里叶变换红外光谱(FTIR)技术,测量了两种不同浓度的枯草芽孢杆菌的红外透过率谱,根据朗伯-比尔定律计算出它们的质量消光截面,通过算出复折射率的虚部,再使用KK(Kramers-Kronig)关系,导出复折射率的实部,并对实验结果作了分析和讨论.通过研究枯草芽孢杆菌复折射率的测量和分析方法,对于进一步研究生物气溶胶的吸收和散射特性、拓宽生物气溶胶的测量和遥测技术方法,具有重要的意义.%The authors measured IR transmission spectra of two different concentrations of bacillus subtilis spores by using Fourier transform infrared spectroscopy (FTIR) technology. The mass extinction cross section k of bacillus subtilis spores was calculated according to Lambert-Beer law and the imaginary part n, of the complex refractive index was also calculated through k. The real part nr of the complex refractive index was derived from the KK (Kramers-Kronig) relationship and the experimental results were also analyzed and discussed with the study of measurement and analysis method of the complex refractive index on bacillus subtilis spores, it is of great significance to further research the absorption and scattering characteristics, and to broaden the measurement and remote sensing technology method of the biological aerosols.

  14. Kinetics of Germination of Individual Bacillus Spores and its Heterogeneity Triggered by Lysozyme%溶菌酶触发的芽孢杆菌芽孢萌发及其异质性研究

    Institute of Scientific and Technical Information of China (English)

    王桂文; 张鹏飞; 王晓春; 陈欢君; Peter Setlow; Li Yongqing

    2016-01-01

    【目的】了解溶菌酶触发芽孢杆菌孢子萌发的异质性及其机制,为认识芽孢(Spore)萌发机制和杀灭芽孢提供参考。【方法】应用拉曼光谱和微分干涉差(DIC)显微镜成像技术高通量分析大量单个Bacillus subtilis (Bs)和B.megaterium (Bm)芽孢经溶菌酶触发的萌发动态。【结果】溶菌酶浓度和温度越高,芽孢萌发越快,孢内CaDPA开始快速释放时间(Tlag )、快速释放所需时间(ΔTrelease )和芽孢皮层水解所需时间(ΔTlys )越短;低于20℃,Bs芽孢萌发的ΔTrelease值是25℃时的4倍以上。SpoVA蛋白高表达菌株的ΔTrelease值和普通菌株基本相同,而缺少皮层水解酶的菌株ΔTrelease值高于普通菌株。95℃处理15 min的孢子,其Tlag、ΔTrelease和ΔTlys值是对照的2倍以上。Bs芽孢萌发的异质性明显,不仅表现在芽孢间,也表现在菌株间。Bm芽孢对溶菌酶更敏感,芽孢间的异质性显著低于Bs。【结论】溶菌酶触发的芽孢萌发在物种、菌株和单细胞层面都存在显著的异质性;溶菌酶浓度和温度对芽孢萌发有重大影响;皮层水解酶也可能参与溶菌酶触发的芽孢萌发进程。%Objective]The heterogeneous germination and its mechanism of Baillus spores trig-gered by lysozyme were studied in order to look insight into the mechanism of spore germina-tion in general and provide new knowledge of inactivating bacterial spores.[Methods]The ki-netic of germinations of multiple individual decoated spores of Bacillus subtilis (Bs)and B. megaterium (Bm)triggered by lysozyme were followed by Raman spectroscopy and differentail interference contrast (DIC)microscopy.[Results]Higher concentrations of lysozyme and tem-peratures significantly speeded the germination of Bs decaoted spores and reduced the time Tlag , at which spores began release of the great major-ity of spores’1∶1 chelate of Ca2+ with dipico-linic acid (Ca

  15. Hydrogen Peroxide-Resistant CotA and YjqC of Bacillus altitudinis Spores Are a Promising Biocatalyst for Catalyzing Reduction of Sinapic Acid and Sinapine in Rapeseed Meal

    Science.gov (United States)

    Zhang, Yanzhou; Li, Xunhang; Hao, Zhikui; Xi, Ruchun; Cai, Yujie; Liao, Xiangru

    2016-01-01

    For the more efficient detoxification of phenolic compounds, a promising avenue would be to develop a multi-enzyme biocatalyst comprising peroxidase, laccase and other oxidases. However, the development of this multi-enzyme biocatalyst is limited by the vulnerability of fungal laccases and peroxidases to hydrogen peroxide (H2O2)-induced inactivation. Therefore, H2O2-resistant peroxidase and laccase should be exploited. In this study, H2O2-stable CotA and YjqC were isolated from the outer coat of Bacillus altitudinis SYBC hb4 spores. In addition to the thermal and alkali stability of catalytic activity, CotA also exhibited a much higher H2O2 tolerance than fungal laccases from Trametes versicolor and Trametes trogii. YjqC is a sporulation-related manganese (Mn) catalase with striking peroxidase activity for sinapic acid (SA) and sinapine (SNP). In contrast to the typical heme-containing peroxidases, the peroxidase activity of YjqC was also highly resistant to inhibition by H2O2 and heat. CotA could also catalyze the oxidation of SA and SNP. CotA had a much higher affinity for SA than B. subtilis CotA. CotA and YjqC rendered from B. altitudinis spores had promising laccase and peroxidase activities for SA and SNP. Specifically, the B. altitudinis spores could be regarded as a multi-enzyme biocatalyst composed of CotA and YjqC. The B. altitudinis spores were efficient for catalyzing the degradation of SA and SNP in rapeseed meal. Moreover, efficiency of the spore-catalyzed degradation of SA and SNP was greatly improved by the presence of 15 mM H2O2. This effect was largely attributed to synergistic biocatalysis of the H2O2-resistant CotA and YjqC toward SA and SNP. PMID:27362423

  16. Hydrogen Peroxide-Resistant CotA and YjqC of Bacillus altitudinis Spores Are a Promising Biocatalyst for Catalyzing Reduction of Sinapic Acid and Sinapine in Rapeseed Meal.

    Directory of Open Access Journals (Sweden)

    Yanzhou Zhang

    Full Text Available For the more efficient detoxification of phenolic compounds, a promising avenue would be to develop a multi-enzyme biocatalyst comprising peroxidase, laccase and other oxidases. However, the development of this multi-enzyme biocatalyst is limited by the vulnerability of fungal laccases and peroxidases to hydrogen peroxide (H2O2-induced inactivation. Therefore, H2O2-resistant peroxidase and laccase should be exploited. In this study, H2O2-stable CotA and YjqC were isolated from the outer coat of Bacillus altitudinis SYBC hb4 spores. In addition to the thermal and alkali stability of catalytic activity, CotA also exhibited a much higher H2O2 tolerance than fungal laccases from Trametes versicolor and Trametes trogii. YjqC is a sporulation-related manganese (Mn catalase with striking peroxidase activity for sinapic acid (SA and sinapine (SNP. In contrast to the typical heme-containing peroxidases, the peroxidase activity of YjqC was also highly resistant to inhibition by H2O2 and heat. CotA could also catalyze the oxidation of SA and SNP. CotA had a much higher affinity for SA than B. subtilis CotA. CotA and YjqC rendered from B. altitudinis spores had promising laccase and peroxidase activities for SA and SNP. Specifically, the B. altitudinis spores could be regarded as a multi-enzyme biocatalyst composed of CotA and YjqC. The B. altitudinis spores were efficient for catalyzing the degradation of SA and SNP in rapeseed meal. Moreover, efficiency of the spore-catalyzed degradation of SA and SNP was greatly improved by the presence of 15 mM H2O2. This effect was largely attributed to synergistic biocatalysis of the H2O2-resistant CotA and YjqC toward SA and SNP.

  17. Genomics, evolution, and crystal structure of a new family of bacterial spore kinases

    OpenAIRE

    2009-01-01

    Bacterial spore formation is a complex process of fundamental relevance to biology and human disease. The spore coat structure is complex and poorly understood, and the roles of many of the protein components remain unclear. We describe a new family of spore coat proteins, the bacterial spore kinases (BSKs), and the first crystal structure of a BSK, YtaA (CotI) from Bacillus subtilis. BSKs are widely distributed in spore-forming Bacillus and Clostridium species, and have a dynamic evolutionar...

  18. Incidence, diversity and characteristics of spores of psychrotolerant spore formers in various REPFEDS produced in Belgium.

    Science.gov (United States)

    Samapundo, S; Devlieghere, F; Xhaferi, R; Heyndrickx, M

    2014-12-01

    The major objectives of this study were to determine the incidence of psychrotolerant spore formers from REPFEDS marketed in Belgium, and their diversity and characteristics. Spore formers in general were found as spores on 38.3% of the food samples and in 85% food products types evaluated. 76% of the food samples containing spore formers had spores before enrichment. A total of 86 spore formers were isolated from the samples. 28 of 86 bacterial spore formers (32.6%) were capable of vegetative growth at 7 °C. 96% (27/28) of these psychrotolerant spore formers were determined to belong to Bacillus or related genera. According to a (GTG)5-PCR analysis, 24 of these 28 isolates were genetically distinct from each other. 10.7% (3/28) of the bacilli were determined to belong to the Bacillus cereus group, namely B. cereus (chicken curry and Edam cheese) and Bacillus mycoides (Emmental cheese). Almost half of the bacilli (12/27) were putatively identified as Bacillus pumilus, which occurs ubiquitously in nature and has been associated with outbreaks of foodborne disease. Only one psychrotolerant clostridium, Clostridium tyrobutyricum, was isolated in the study. The results of this study show the highly diverse ecology and spoilage potential of psychrotolerant spore formers in REPFEDs marketed in Belgium.

  19. Production and stability of chlorine dioxide in organic acid solutions as affected by pH, type of acid, and concentration of sodium chlorite, and its effectiveness in inactivating Bacillus cereus spores.

    Science.gov (United States)

    Kim, Hoikyung; Kang, Youngjee; Beuchat, Larry R; Ryu, Jee-Hoon

    2008-12-01

    We studied the production and stability of chlorine dioxide (ClO(2)) in organic acid solutions and its effectiveness in killing Bacillus cereus spores. Sodium chlorite (5000, 10,000, or 50,000 microg/ml) was added to 5% acetic, citric, or lactic acid solution, adjusted to pH 3.0, 4.0, 5.0, or 6.0, and held at 21 degrees C for up to 14 days. The amount of ClO(2) produced was higher as the concentration of sodium chlorite was increased and as the pH of the acid solutions was decreased. However, the stability in production of ClO(2) was enhanced by increasing the pH of the organic acid solutions. To evaluate the lethal activity of ClO(2) produced in various acid solutions as affected by acidulant and pH, suspensions of B. cereus spores were treated at 21 degrees C for 1, 3, 5, or 10 min in hydrochloric acid or organic acid solutions (pH 3.0, 4.0, 5.0, or 6.0) containing ClO(2) at concentrations of 100, 50, or 25 microg/ml. Populations of viable spores treated with ClO(2) at concentrations of 100 or 50 microg/ml in organic acid solutions decreased more rapidly than populations treated with the same concentrations of ClO(2) in HCl. Rates of inactivation tended to increase with higher pH of ClO(2) solutions. Results show that ClO(2) formed in organic acid solutions has higher stability and is more lethal to B. cereus spores than ClO(2) formed at the same concentration in HCl solution. This finding emphasizes the benefits of using organic acid solutions to prepare ClO(2) intended for use as an antimicrobial.

  20. Detecting Cortex Fragments During Bacterial Spore Germination.

    Science.gov (United States)

    Francis, Michael B; Sorg, Joseph A

    2016-06-25

    The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

  1. Bacterial spores in food : how phenotypic variability complicates prediction of spore properties and bacterial behavior

    NARCIS (Netherlands)

    Eijlander, Robyn T.; Abee, Tjakko; Kuipers, Oscar P.

    2011-01-01

    Bacillus spores are a known cause of food spoilage and their increased resistance poses a major challenge in efficient elimination. Recent studies on bacterial cultures at the single cell level have revealed how minor differences in essential spore properties, such as core water content or germinant

  2. Bacterial spores in food: how phenotypic variability complicates prediction of spore properties and bacterial behavior

    NARCIS (Netherlands)

    Eijlander, R.T.; Abee, T.; Kuipers, O.P.

    2011-01-01

    Bacillus spores are a known cause of food spoilage and their increased resistance poses a major challenge in efficient elimination. Recent studies on bacterial cultures at the single cell level have revealed how minor differences in essential spore properties, such as core water content or germinant

  3. Application of spore laccase from Bacillus amyloliquefaciens in dye decolorization%解淀粉芽孢杆菌芽孢漆酶在染料脱色中的应用

    Institute of Scientific and Technical Information of China (English)

    栗君; 李国富; 芦磊; 潘俊波; 赵敏; 王天女; 徐腾飞; 王靖瑶

    2013-01-01

    Spore laccase from Bacillus amyloliquefaciens LC03 has good stability. In this work, spore suspension with laccase activity was prepared and tested for its ability in decolorization of four synthetic dyes,including remazol brilliant blue R(RBBR) , reactive black 5, indigo carmine and crystal violet. Laccase mediators were screened to improve the decolorization process. The effects of enzyme and mediator concentration on simulated dye effluent decolorization were also investigated. The results showed that no decolorization of RBBR, reactive black 5 and indigo carmine occurred in the absence of mediator, while more than 60. 00% decolorization of the three dyes were obtained when acetosyringone ( ACE) was present. The spore laccase-mediator system could efficiently decolorize the simulated dye effluent with 88. 64 U/L of spore laccase and 0. 5 mmol/L of ACE at pH 9. 0. More than 80. 00% of simulated dye effluent was decolorized after 2 hours, indicating potential application of this spore laccase in dye wastewater treatment.%解淀粉芽孢杆菌LC03的芽孢漆酶具有较好的稳定性,通过制备具有漆酶活性的芽孢悬液,研究了芽孢漆酶对4种合成染料RB亮蓝、活性黑、靛红和结晶紫的脱色效果,并筛选出促进染料脱色的漆酶介体,同时考察了酶浓度、介体浓度对模拟染料废水脱色的影响.结果表明:RB亮蓝、活性黑和靛红在无介体时不能被脱色,在乙酰丁香酮(ACE)介导下的脱色率都超过了60.00%;在pH9.0时,芽孢漆酶-介体系统对模拟染料废水脱色的酶浓度和介体浓度分别为88.64 U/L和0.5 mmol/L时,2h后脱色率可超过80.00%,表明该芽孢漆酶在染料废水的处理上具有较好的应用前景.

  4. SELEX法筛选炭疽芽孢杆菌芽孢适配子的研究%In vitro selection of DNA aptamers to Bacillus anthracis spores by SELEX

    Institute of Scientific and Technical Information of China (English)

    甄蓓; 宋亚军; 郭兆彪; 王津; 张敏丽; 俞守义; 杨瑞馥

    2001-01-01

    Objective:To obtain oligonucleotide aptamers which can specifically bind to Bacillus anthracis spores by in vitro selection protocol-SELEX (system evolution of ligands by exponential enrichment).Methods:An in vitro synthesized 78 mer random DNA library (≤1014-15types of different DNAs ) was subjected to 15 rounds of selection using SELEX method against spores of B.anthracis vaccine strain A.16R. Binding of the aptamers to spores was visualized by biotin-streptavidin-horseradish peroxidase system.Results:PCR amplification band pattern of the first round selection was different from that of the ninth round. The binding assay demonstrated that D absorbance at 450 nm of the fifteenth round pool increased 9 times as compared with that of the first round , and the D absorbance increased with the increment of aptamers′ quantity binding to spores. Conclusions: A set of aptamers with considerable binding affinity to B.anthracis spores were successfully selected from the initial random DNA pool.%目的:利用SELEX(system evolution of ligands by exponential enrichment)体外筛选技术,寻找能与炭疽芽孢杆菌芽孢特异结合的寡核苷酸适配子(aptamer).方法:体外合成长度为78个核苷酸的随机DNA库,通过SELEX技术,以炭疽芽孢杆菌疫苗株A.16R的芽孢为靶标进行15轮的筛选,利用生物素-亲和素显色系统判断寡核苷酸与芽孢的结合活性.结果:随着筛选轮数的增加,PCR扩增电泳条带逐渐单纯,寡核苷酸与芽孢结合后显色,D值提高了9倍以上,D值随适配子结合量的增加而递增.结论:已初步筛选到与炭疽芽孢具有亲和力的适配子.

  5. Inside the Meteorite — Bacterial Spore Survival After Exposure to Galactic Cosmic Radiation

    Science.gov (United States)

    Moeller, R.; Berger, T.; Matthiä, D.; Okayasu, R.; Kato, T.; Kitamura, H.; Reitz, G.

    2010-04-01

    Based on their unique resistance to various space parameters, bacterial spores are one of the model systems used for astrobiological studies. In our research, we studied the response of Bacillus subtilis spores to the exposure of galactic cosmic radiation.

  6. Inactivation of chemical and heat-resistant spores of Bacillus and Geobacillus by nitrogen cold atmospheric plasma and comparison to thermal and chemical based methods

    NARCIS (Netherlands)

    Bokhorst-van de Veen, van H.; Xie, H.; Esveld, D.C.; Abee, T.; Mastwijk, H.C.; Nierop Groot, M.N.

    2015-01-01

    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 s

  7. Biosorption of uranium and copper by Bacillus Sphaericus JG-A12 cells, spores and S-layer proteins embedded in sol-gel ceramics

    Energy Technology Data Exchange (ETDEWEB)

    Raff, J.; Soltmann, U.; Boettcher, H. [Arbeitsgruppe Funktionelle Schichten, GMBU e.V., Dresden (Germany); Matys, S.; Pompe, W. [Inst. fuer Materialwissenschaft, Technische Univ. Dresden (Germany); Selenska-Pobell, S.

    2002-05-01

    Vegetative cells, spores and stabilized S-layer sheets of B. sphaericus JG-A12 were embedded in SiO{sub 2} bulk particles using sol-gel techniques. In sorption experiments the metal binding capacity of the free biocomponents and the corresponding biological ceramics were compared. (orig.)

  8. Identification of CdnL, a putative transcriptional regulator involved in repair and outgrowth of heat-damaged bacillus cereus spores

    NARCIS (Netherlands)

    Warda, Alicja K.; Tempelaars, Marcel H.; Boekhorst, Jos; Abee, Tjakko; Groot, Masja Nierop N.

    2016-01-01

    Spores are widely present in the environment and are common contaminants in the food chain, creating a challenge for food industry. Nowadays, heat treatments conventionally applied in food processing may become milder to comply with consumer desire for products with higher sensory and nutritional

  9. Toxicological Evaluation of a Potential Immunosensitizer for Use as a Mucosal Adjuvant—Bacillus thuringiensis Cry1Ac Spore-Crystals: A Possible Inverse Agonist that Deserves Further Investigation

    Directory of Open Access Journals (Sweden)

    Bélin Poletto Mezzomo

    2015-12-01

    Full Text Available In addition to their applicability as biopesticides, Bacillus thuringiensis (Bt Cry1Ac spore-crystals are being researched in the immunology field for their potential as adjuvants in mucosal and parenteral immunizations. We aimed to investigate the hematotoxicity and genotoxicity of Bt spore-crystals genetically modified to express Cry1Ac individually, administered orally (p.o. or with a single intraperitoneal (i.p. injection 24 h before euthanasia, to simulate the routes of mucosal and parenteral immunizations in Swiss mice. Blood samples were used to perform hemogram, and bone marrow was used for the micronucleus test. Cry1Ac presented cytotoxic effects on erythroid lineage in both routes, being more severe in the i.p. route, which also showed genotoxic effects. The greater severity noted in this route, mainly at 6.75 mg/kg, as well as the intermediate effects at 13.5 mg/kg, and the very low hematotoxicity at 27 mg/kg, suggested a possible inverse agonism. The higher immunogenicity for the p.o. route, particularly at 27 mg/kg, suggested that at this dose, Cry 1Ac could potentially be used as a mucosal adjuvant (but not in parenteral immunizations, due to the genotoxic effects observed. This potential should be investigated further, including making an evaluation of the proposed inverse agonism and carrying out cytokine profiling.

  10. Bacterial spore germination and protein mobility.

    Science.gov (United States)

    Moir, Anne

    2003-10-01

    Fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) has been used to report on protein mobility in single spores. Proteins found in dormant Bacillus spores are not mobile; however, mobility is restored when germination occurs and the core rehydrates. Spores of a cwlD mutant, in which the cortex is resistant to hydrolysis, are able to complete the earliest stages of germination in response to a specific germinant stimulus; in these circumstances, the protein in the spore remains immobile. Therefore, the earliest stages of spore germination, including loss of resistance to extreme heat and the complete release of the spore component dipicolinic acid, are achieved without the restoration of protein mobility.

  11. Response in soil of Cupriavidus necator and other copper-resistant bacterial predators of bacteria to addition of water, soluble nutrients, various bacterial species, or Bacillus thuringiensis spores and crystals

    Energy Technology Data Exchange (ETDEWEB)

    Casida, L.E. Jr. (Pennsylvania State Univ., University Park (USA))

    1988-09-01

    Soil was incubated with various species of bacteria, Bacillus subtilis, or Bacillus thuringiensis spores and crystals. These were added to serve as potential prey for indigenous, copper-resistant, nonobligate bacterial predators of bacteria in the soil. Alternatively, the soil was incubated with soluble nutrients or water only to cause potential indigenous prey cells to multiply so the predator cells would multiply. All of these incubation procedures caused excessive multiplication of some gram-negative bacteria in soil. Even greater multiplication, however, often occurred for certain copper-resistant bacterial predators of bacteria that made up a part of the gram-negative response. Incubation of the soil with copper per se did not give these responses. In most cases, the copper-resistant bacteria that responded were Cupriavidus necator, bacterial predator L-2, or previously unknown bacteria that resembled them. The results suggest that, under various conditions of soil incubation, gram-negative bacterial predators of bacteria multiply and that several copper-resistant types among them can be detected, counted, and isolated by plating dilutions of the soil onto media containing excess copper.

  12. [Distribution of strains of spore-forming bacteria of the genus Bacillus in the bottom sediments of Lake Khubsugul in Northern Mongolia as an indication of paleoclimate].

    Science.gov (United States)

    Suslova, M Iu; Parfenova, V V; Ziborova, G A; Fedotov, A P

    2009-01-01

    Data on the distribution and abundance of bacteria of the genus Bacillus in the bottom sediments of Lake Khubsugul have shown the predominance of strains that preferred low temperatures. This indicates fairly cold temperature conditions on the territory of the Khubsugul drainage area. On the whole, the dynamics of interchange of minimums and maximums of abundance of bacteria of the genus Bacillus is similar to the global climate fluctuations. Study of the enzymatic activity of pure cultures revealed that most strains studied possessed proteolytic activity; consequently, the dynamics of bacteria development is correlated with the supply of organic nitrogen-containing compositions.

  13. Cortex Peptidoglycan Lytic Activity in Germinating Bacillus anthracis Spores▿

    OpenAIRE

    2008-01-01

    Bacterial endospore dormancy and resistance properties depend on the relative dehydration of the spore core, which is maintained by the spore membrane and its surrounding cortex peptidoglycan wall. During spore germination, the cortex peptidoglycan is rapidly hydrolyzed by lytic enzymes packaged into the dormant spore. The peptidoglycan structures in both dormant and germinating Bacillus anthracis Sterne spores were analyzed. The B. anthracis dormant spore peptidoglycan was similar to that fo...

  14. The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host.

    Science.gov (United States)

    Stewart, George C

    2015-12-01

    Much of what we know regarding bacterial spore structure and function has been learned from studies of the genetically well-characterized bacterium Bacillus subtilis. Molecular aspects of spore structure, assembly, and function are well defined. However, certain bacteria produce spores with an outer spore layer, the exosporium, which is not present on B. subtilis spores. Our understanding of the composition and biological functions of the exosporium layer is much more limited than that of other aspects of the spore. Because the bacterial spore surface is important for the spore's interactions with the environment, as well as being the site of interaction of the spore with the host's innate immune system in the case of spore-forming bacterial pathogens, the exosporium is worthy of continued investigation. Recent exosporium studies have focused largely on members of the Bacillus cereus family, principally Bacillus anthracis and Bacillus cereus. Our understanding of the composition of the exosporium, the pathway of its assembly, and its role in spore biology is now coming into sharper focus. This review expands on a 2007 review of spore surface layers which provided an excellent conceptual framework of exosporium structure and function (A. O. Henriques and C. P. Moran, Jr., Annu Rev Microbiol 61:555-588, 2007, http://dx.doi.org/10.1146/annurev.micro.61.080706.093224). That review began a process of considering outer spore layers as an integrated, multilayered structure rather than simply regarding the outer spore components as independent parts.

  15. The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

    OpenAIRE

    2015-01-01

    Much of what we know regarding bacterial spore structure and function has been learned from studies of the genetically well-characterized bacterium Bacillus subtilis. Molecular aspects of spore structure, assembly, and function are well defined. However, certain bacteria produce spores with an outer spore layer, the exosporium, which is not present on B. subtilis spores. Our understanding of the composition and biological functions of the exosporium layer is much more limited than that of oth...

  16. Research on Ozone Inactivation of Bacillus subtilis Spores%臭氧对枯草芽孢杆菌孢子的灭活研究

    Institute of Scientific and Technical Information of China (English)

    齐爱玲; 李继; 纪家林; 邹俐; 郭路路

    2011-01-01

    Cryptosporidium parvum oocysts bring challenges to drinking water safety due to their high resistance to the disinfectants, resulting in great potential harm to public health. This research rehtes to B. Subtilis spores(ATCC 6633)used as a surrogate microorganism to study the impact of turbidity and other water parameters as well as ozone concentration on the inactivation effect. The result showed that ozone inactivation of B. Subtilis spores was mainly associated with CT value rather than the concentration of ozone per se, and reduction of turbidity and organic matter in water increased inactivation efficiency. In addition, when water temperature became low it was necessary to increase CT value to ensure the disinfection.%隐孢子虫灭活困难,给饮用水安全带来了挑战.实验以枯草芽孢杆菌孢子(ATCC6633)作为隐孢子虫的指示菌,研究了臭氧浓度、浊度、有机物、温度等因素对臭氧灭活枯草芽孢杆菌孢子的影响.研究表明,臭氧对枯草芽孢杆菌孢子的灭活与CT值相关,臭氧浓度对消毒效果影响较小;降低浊度、有机物含量,能提高臭氧对枯草芽孢杆菌孢子的灭活效率;温度降低,为保证一定的消毒效率,所需的CT值越大.研究结果为水厂合理控制运行参数提供了借鉴.

  17. The Molecular Timeline of a Reviving Bacterial Spore

    OpenAIRE

    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 trans...

  18. Relevance of diffusion through bacterial spore coats/membranes and the associated concentration boundary layers in the initial lag phase of inactivation: a case study for Bacillus subtilis with ozone and monochloramine.

    Science.gov (United States)

    Fernando, W J N; Othman, R

    2006-02-01

    Disinfectants are generally used to inactivate microorganisms in solutions. The process of inactivation involves the disinfectant in the liquid diffusing towards the bacteria sites and thereafter reacting with bacteria at rates determined by the respective reaction rates. Such processes have demonstrated an initial lag phase followed by an active depletion phase of bacteria. This paper attempts to study the importance of the combined effects of diffusion of the disinfectant through the outer membrane of the bacteria and transport through the associated concentration boundary layers (CBLs) during the initial lag phase. Mathematical equations are developed correlating the initial concentration of the disinfectant with time required for reaching a critical concentration (C*) at the inner side of the membrane of the cell based on diffusion of disinfectant through the outer membranes of the bacteria and the formation of concentration boundary layers on both sides of the membranes. Experimental data of the lag phases of inactivation already available in the literature for inactivation of Bacillus subtilis spores with ozone and monochloramine are tested with the equations. The results seem to be in good agreement with the theoretical equations indicating the importance of diffusion process across the outer cell membranes and the resulting CBL's during the lag phase of disinfection.

  19. Effect of modified atmosphere and temperature abuse on the growth from spores and cereulide production of Bacillus weihenstephanensis in a cooked chilled meat sausage

    DEFF Research Database (Denmark)

    Thorsen, Line; Budde, Birgitte Bjørn; Koch, Anette Granly;

    2009-01-01

    demonstrates that MAP can be used to inhibit growth of a psychrotolerant toxin producing Bacillus spp. during chill storage at 8 °C, and substantially reduce the risk of emetic food poisoning at abuse condition. Results are of relevance for improving safety of ready to eat processed chilled foods of extended...... and temperature abuse for 1.5 h daily at 20 °C during 1 week resulted in increased cell counts and variable cereulide production in the meat sausage. A pre-history at 8 °C for 1 week inMAP with OTR of 1.3 or 40ml/m2/24 h and 2% O2 resulted in cereulide concentrations of 0.816-1.353 µg/gmeat sausage, while a pre-history...

  20. Mechanisms of Resistance in Microbial Spores

    Science.gov (United States)

    1990-12-20

    solids (and water) content by immersion refractometry . Heat-activated spores of Bacillus stearotherrnophilus were found to be separable into two...incrC· ment of bacterial cells, enabling determination of their solids content by immersion refractometry . The results agreed well with values for

  1. Bacillus probiotics.

    Science.gov (United States)

    Cutting, Simon M

    2011-04-01

    Bacterial spore formers are being used as probiotic supplements for use in animal feeds, for human dietary supplements as well as in registered medicines. Their heat stability and ability to survive the gastric barrier makes them attractive as food additives and this use is now being taken forward. While often considered soil organisms this conception is misplaced and Bacilli should be considered as gut commensals. This review summarises the current use of Bacillus species as probiotics, their safety, mode of action as well as their commercial applications.

  2. Maximum shields: the assembly and function of the bacterial spore coat.

    Science.gov (United States)

    Driks, Adam

    2002-06-01

    Spores produced by bacilli and clostridia are surrounded by a multilayered protein shell called the coat. As the armor-like appearance of the coat suggests, this structure, along with others within the spore, confers the remarkable resistance properties that make Bacillus anthracis spores such potent biological weapons. Here, I review recent studies of coat assembly in the model organism Bacillus subtilis, and explore the implications of these findings for coat assembly in B. anthracis and for defense against biological weapons.

  3. Contamination of healthcare workers' hands with bacterial spores.

    Science.gov (United States)

    Sasahara, Teppei; Ae, Ryusuke; Watanabe, Michiyo; Kimura, Yumiko; Yonekawa, Chikara; Hayashi, Shunji; Morisawa, Yuji

    2016-08-01

    Clostridium species and Bacillus spp. are spore-forming bacteria that cause hospital infections. The spores from these bacteria are transmitted from patient to patient via healthcare workers' hands. Although alcohol-based hand rubbing is an important hand hygiene practice, it is ineffective against bacterial spores. Therefore, healthcare workers should wash their hands with soap when they are contaminated with spores. However, the extent of health care worker hand contamination remains unclear. The aim of this study is to determine the level of bacterial spore contamination on healthcare workers' hands. The hands of 71 healthcare workers were evaluated for bacterial spore contamination. Spores attached to subject's hands were quantitatively examined after 9 working hours. The relationship between bacterial spore contamination and hand hygiene behaviors was also analyzed. Bacterial spores were detected on the hands of 54 subjects (76.1%). The mean number of spores detected was 468.3 CFU/hand (maximum: 3300 CFU/hand). Thirty-seven (52.1%) and 36 (50.7%) subjects were contaminated with Bacillus subtilis and Bacillus cereus, respectively. Nineteen subjects (26.8%) were contaminated with both Bacillus species. Clostridium difficile was detected on only one subject's hands. There was a significant negative correlation between the hand contamination level and the frequency of handwashing (r = -0.44, P < 0.01) and a significant positive correlation between the hand contamination level and the elapsed time since last handwashing (r = 0.34, P < 0.01). Healthcare workers' hands may be frequently contaminated with bacterial spores due to insufficient handwashing during daily patient care.

  4. Bacterial spore structures and their protective role in biocide resistance.

    Science.gov (United States)

    Leggett, M J; McDonnell, G; Denyer, S P; Setlow, P; Maillard, J-Y

    2012-09-01

    The structure and chemical composition of bacterial spores differ considerably from those of vegetative cells. These differences largely account for the unique resistance properties of the spore to environmental stresses, including disinfectants and sterilants, resulting in the emergence of spore-forming bacteria such as Clostridium difficile as major hospital pathogens. Although there has been considerable work investigating the mechanisms of action of many sporicidal biocides against Bacillus subtilis spores, there is far less information available for other species and particularly for various Clostridia. This paucity of information represents a major gap in our knowledge given the importance of Clostridia as human pathogens. This review considers the main spore structures, highlighting their relevance to spore resistance properties and detailing their chemical composition, with a particular emphasis on the differences between various spore formers. Such information will be vital for the rational design and development of novel sporicidal chemistries with enhanced activity in the future.

  5. [Bacterial spore--a new vaccine vehicle--a review].

    Science.gov (United States)

    Wang, Yanchun; Zhang, Zhaoshan

    2008-03-01

    Bacterial spores are robust and dormant life forms with formidable resistance properties. Spores of the genus Bacillus have been used for a long time as probiotics for oral bacteriotherapy both in humans and animals. Recently, genetically modified B. subtilis spores and B. anthracis spores have been used as indestructible delivery vehicles for vaccine antigens. They were used as vaccine vehicles or spore vaccine for oral immunization against tetanus and anthrax, and the results were very exciting. Unlike many second generation vaccine systems currently under development, bacterial spores offer heat stability and the flexibility for genetic manipulation. At the same time, they can elicit mucosal immune response by oral and nasal administration. This review focuses on the use of recombinant spores as vaccine delivery vehicles.

  6. Imaging bacterial spores by soft-x-ray microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Stead, A.D.; Ford, T.W. [Univ. of London, Surrey (United Kingdom); Judge, J. [Unilever plc, Sharnbrook (United Kingdom)] [and others

    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 by 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.

  7. Initiation of bacterial spore germination.

    Science.gov (United States)

    Vary, J C; Halvorson, H O

    1968-04-01

    To investigate the problem of initiation in bacterial spore germination, we isolated, from extracts of dormant spores of Bacillus cereus strain T and B. licheniformis, a protein that initiated spore germination when added to a suspension of heat-activated spores. The optimal conditions for initiatory activity of this protein (the initiator) were 30 C in 0.01 to 0.04 m NaCl and 0.01 m tris(hydroxymethyl)aminomethane (pH 8.5). The initiator was inhibited by phosphate but required two co-factors, l-alanine (1/7 of K(m) for l-alanine-inhibited germination) and nicotinamide adenine dinucleotide (1.25 x 10(-4)m). In the crude extract, the initiator activity was increased 3.5-fold by heating the extract at 65 C for 10 min, but the partially purified initiator preparation was completely heat-sensitive (65 C for 5 min). Heat stability could be conferred on the purified initiator by adding 10(-3)m dipicolinic acid. A fractionation of this protein that excluded l-alanine dehydrogenase and adenosine deaminase from the initiator activity was developed. The molecular weight of the initiator was estimated as 7 x 10(4). The kinetics of germination in the presence of initiator were examined at various concentrations of l-alanine and nicotinamide adenine dinucleotide.

  8. Evaluation of up-converting phosphor technology-based lateral flow strips for rapid detection of Bacillus anthracis Spore, Brucella spp., and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Pingping Zhang

    Full Text Available Bacillus anthracis, Brucella spp., and Yersinia pestis are zoonotic pathogens and biowarfare- or bioterrorism-associated agents that must be detected rapidly on-site from various samples (e.g., viscera and powders. An up-converting phosphor technology-based lateral flow (UPT-LF strip was developed as a point-of-care testing (POCT to satisfy the requirements of first-level emergency response. We developed UPT-LF POCT to quantitatively detect the three pathogens within 15 min. Sample and operation-error tolerances of the assay were comprehensively evaluated. The sensitivity of UPT-LF assay to bacterial detection reached 10(4 cfu · mL(-1 (100 cfu/test, with a linear quantitative range of 4 to 6 orders of magnitude. Results revealed that the UPT-LF assay exhibited a high specificity with the absence of false-positive results even at 10(9 cfu · mL(-1 of non-specific bacterial contamination. The assay could tolerate samples with a wide pH range (2 to 12, high ion strengths (≥ 4 mol · L(-1 of NaCl, high viscosities (≤ 25 mg · mL(-1 of PEG20000 or ≥ 20% of glycerol, and high concentrations of bio-macromolecule (≤ 200 mg · mL(-1 of bovine serum albumin or ≥ 80 mg · mL(-1 of casein. The influence of various types of powders and viscera (fresh and decomposed on the performance of UPT-LF assay was determined. The operational error of liquid measurement exhibited few effects on sensitivity and specificity. The developed UPT-LF POCT assay is applicable under field conditions with excellent tolerance to sample complexity and operational error.

  9. Comparing the mannitol-egg yolk-polymyxin agar plating method with the three-tube most-probable-number method for enumeration of Bacillus cereus spores in raw and high-temperature, short-time pasteurized milk.

    Science.gov (United States)

    Harper, Nigel M; Getty, Kelly J K; Schmidt, Karen A; Nutsch, Abbey L; Linton, Richard H

    2011-03-01

    The U.S. Food and Drug Administration's Bacteriological Analytical Manual recommends two enumeration methods for Bacillus cereus: (i) standard plate count method with mannitol-egg yolk-polymyxin (MYP) agar and (ii) a most-probable-number (MPN) method with tryptic soy broth (TSB) supplemented with 0.1% polymyxin sulfate. This study compared the effectiveness of MYP and MPN methods for detecting and enumerating B. cereus in raw and high-temperature, short-time pasteurized skim (0.5%), 2%, and whole (3.5%) bovine milk stored at 4°C for 96 h. Each milk sample was inoculated with B. cereus EZ-Spores and sampled at 0, 48, and 96 h after inoculation. There were no differences (P > 0.05) in B. cereus populations among sampling times for all milk types, so data were pooled to obtain overall mean values for each treatment. The overall B. cereus population mean of pooled sampling times for the MPN method (2.59 log CFU/ml) was greater (P plate count method (1.89 log CFU/ml). B. cereus populations in the inoculated milk samples ranged from 2.36 to 3.46 and 2.66 to 3.58 log CFU/ml for inoculated milk treatments for the MYP plate count and MPN methods, respectively, which is below the level necessary for toxin production. The MPN method recovered more B. cereus, which makes it useful for validation research. However, the MYP plate count method for enumeration of B. cereus also had advantages, including its ease of use and faster time to results (2 versus 5 days for MPN).

  10. Characterization of Bacillus anthracis persistence in vivo.

    Directory of Open Access Journals (Sweden)

    Sarah A Jenkins

    Full Text Available Pulmonary exposure to Bacillus anthracis spores initiates inhalational anthrax, a life-threatening infection. It is known that dormant spores can be recovered from the lungs of infected animals months after the initial spore exposure. Consequently, a 60-day course antibiotic treatment is recommended for exposed individuals. However, there has been little information regarding details or mechanisms of spore persistence in vivo. In this study, we investigated spore persistence in a mouse model. The results indicated that weeks after intranasal inoculation with B. anthracis spores, substantial amounts of spores could be recovered from the mouse lung. Moreover, spores of B. anthracis were significantly better at persisting in the lung than spores of a non-pathogenic Bacillus subtilis strain. The majority of B. anthracis spores in the lung were tightly associated with the lung tissue, as they could not be readily removed by lavage. Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium. Confocal analysis indicated that some of the spores were inside epithelial cells. This was further confirmed by differential immunofluorescence staining of lung cells harvested from the infected lungs, suggesting that association with lung epithelial cells may provide an advantage to spore persistence in the lung. There was no or very mild inflammation in the infected lungs. Furthermore, spores were present in the lung tissue as single spores rather than in clusters. We also showed that the anthrax toxins did not play a role in persistence. Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.

  11. Effects of the dietary supplementation of mixed probiotic spores of Bacillus amyloliquefaciens 54A, and Bacillus pumilus 47B on growth, innate immunity and stress responses of striped catfish (Pangasianodon hypophthalmus).

    Science.gov (United States)

    Truong Thy, Ho Thi; Tri, Nguyen Nhu; Quy, Ong Moc; Fotedar, Ravi; Kannika, Korntip; Unajak, Sasimanas; Areechon, Nontawith

    2017-01-01

    The study used the mixed probiotics of Bacillus amyloliquefaciens 54A and B. pumilus 47B isolated from striped catfish (Pangasianodon hypophthalmus) intestine aiming to stimulate growth performance, innate immunity, stress tolerance of striped catfish. The average weight gain (AWG), specific growth rate (SGR), and feed conversion ratio (FCR) were analyzed after fish were fed the mixture of probiotics (B. amyloliquefaciens 54A and B. pumilus 47B) at concentrations of 1 × 10(8), 3 × 10(8), and 5 × 10(8) CFU g(-1) feed for 90 days. Immunity parameters, survival rate of fish challenged with Edwardsiella ictaluri and ammonia tolerance were also investigated. The amounts of B. amyloliquefaciens and B. pumilus were counted and identified by specific primer pairs of Ba1-F/Ba1-R, and 16-F/Bpu-R to confirm the presence of probiotics in fish intestine. The AWG (476.6 ± 7.81 g fish(-1)) of fish fed probiotics at 5 × 10(8) CFU g(-1) was significant higher than the control (390 ± 25.7 g fish(-1)) after 90 days of feeding, but there was no significant (P > 0.05) effect of probiotics on FCR and SGR. Fish fed diet containing probiotics at 5 × 10(8) CFU g(-1) also expressed resistance to E. ictaluri infection and higher immune parameters such as phagocytic activity, respiratory bursts, and lysozyme activity than the control. Stress response with ammonia showed significantly lower mortality rate (25%, 20% and 27%) of fish fed probiotics at all three levels of 1, 3 and 5 × 10(8) CFU g(-1) than the fish fed control diet (75%). The study also demonstrated that the probiotics survived in the intestine of striped catfish after 90 days of feeding. Therefore, the dietary supplementation of a mixture of B. amyloliquefaciens and B. pumilus at 5 × 10(8) CFU g(-1) can be used to improve the health and growth rate of striped catfish.

  12. Chemical and morphological studies of bacterial spore formation. IV. The development of spore refractility.

    Science.gov (United States)

    YOUNG, I E; JAMES, P C

    1962-01-01

    From the stage of a completed membranous forespore to that of a fully ripened free spore, synchronously sporulating cells of a variant Bacillus cereus were studied by cytological and chemical methods. Particular attention was paid to the development of the three spore layers-cortex, coat, and exosporium-in relation to the forespore membrane. First, the cortex is laid down between the recently described (5) double layers of the forespore membrane. Then when the cortex is (1/3) fully formed, the spore coat and exosporium are laid down peripheral to the outer membrane layer covering the cortex. As these latter layers appear, the spores, previously dense by dark phase contrast, gradually "whiten" or show an increase in refractive index. With this whitening, calcium uptake commences, closely followed by the synthesis of dipicolinic acid and the process is terminated, an hour later, with the formation of a fully refractile spore. In calcium-deficient media, final refractility is lessened and dipicolinic acid is formed only in amounts proportional to the available calcium. If calcium is withheld during the period of uptake beyond a critical point, sporulating cells lose the ability to assimilate calcium and to form normal amounts of dipicolinic acid. The resulting deficient spores are liberated from the sporangia but are unstable in water suspensions. Unlike ripe spores, they do not react violently to acid hydrolysis and, in thin sections, their cytoplasmic granules continue to stain with lead solutions.

  13. Effects of Chlorine Dioxide on Spore Structural and Fuctional Properties

    Science.gov (United States)

    2006-05-31

    A., Price, B., Leighton, T. and K. Wheeler. 2003. Kinetics of size changes of individual Bacillus thuringiensis spores in response to changes in...vegetative growth . The germination process involves a defined temporal order of events, characterized initially by hydrolysis of the spore coat and...capable of early germination but not resumption of vegetative growth and cell division. We have explored the use of rapid spectrophotometric assays to

  14. Rapid onsite assessment of spore viability.

    Energy Technology Data Exchange (ETDEWEB)

    Branda, Steven; Lane, Todd W.; VanderNoot, Victoria A.; Gaucher, Sara P.; Jokerst, Amanda S.

    2005-12-01

    This one year LDRD addresses problems of threat assessment and restoration of facilities following a bioterror incident like the incident that closed down mail facilities in late 2001. Facilities that are contaminated with pathogenic spores such as B. anthracis spores must be shut down while they are treated with a sporicidal agent and the effectiveness of the treatment is ascertained. This process involves measuring the viability of spore test strips, laid out in a grid throughout the facility; the CDC accepted methodologies require transporting the samples to a laboratory and carrying out a 48 hr outgrowth experiment. We proposed developing a technique that will ultimately lead to a fieldable microfluidic device that can rapidly assess (ideally less than 30 min) spore viability and effectiveness of sporicidal treatment, returning facilities to use in hours not days. The proposed method will determine viability of spores by detecting early protein synthesis after chemical germination. During this year, we established the feasibility of this approach and gathered preliminary results that should fuel a future more comprehensive effort. Such a proposal is currently under review with the NIH. Proteomic signatures of Bacillus spores and vegetative cells were assessed by both slab gel electrophoresis as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection. The conditions for germination using a number of chemical germinants were evaluated and optimized and the time course of protein synthesis was ascertained. Microseparations were carried out using both viable spores and spores inactivated by two different methods. A select number of the early synthesis proteins were digested into peptides for analysis by mass spectrometry.

  15. Pulling the trigger: the mechanism of bacterial spore germination.

    Science.gov (United States)

    Foster, S J; Johnstone, K

    1990-01-01

    In spite of displaying the most extreme dormancy and resistance properties known among living systems, bacterial endospores retain an alert environment-sensing mechanism that can respond within seconds to the presence of specific germinants. This germination response is triggered in the absence of both germinant and germinant-stimulated metabolism. Genes coding for components of the sensing mechanism in spores of Bacillus subtilis have been cloned and sequenced. However, the molecular mechanism whereby these receptors interact with germinants to initiate the germination response is unknown. Recent evidence has suggested that in spores of Bacillus megaterium KM, proteolytic activation of an autolytic enzyme constitutes part of the germination trigger reaction.

  16. Modeling of Bacillus spores: Inactivation and Outgrowth

    Science.gov (United States)

    2011-03-01

    52 Michaelis - Menten Kinetics ...of repair mechanism [36]. These models were based on Michaelis - Menten kinetics , which is also the foundation of the work in this research Michaelis ...catalyzed reactions. Michaelis - Menten kinetics is a model of enzyme kinetics . The Michaelis - Menten equation describes the rates of enzymatic reactions by

  17. Evaluation of Sampling Methods for Bacillus Spore ...

    Science.gov (United States)

    Journal Article Following a wide area release of biological materials, mapping the extent of contamination is essential for orderly response and decontamination operations. HVAC filters process large volumes of air and therefore collect highly representative particulate samples in buildings. HVAC filter extraction may have great utility in rapidly estimating the extent of building contamination following a large-scale incident. However, until now, no studies have been conducted comparing the two most appropriate sampling approaches for HVAC filter materials: direct extraction and vacuum-based sampling.

  18. Morphogenesis of the Bacillus anthracis Spore

    Science.gov (United States)

    2007-02-01

    Fritsch, and T. Maniatis . 1989. Molecular cloning : a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 72. Santo...Sciences, Illinois Institute of Technology, Chicago, Illinois 606163; Department of Molecular , Cellular, and Developmental Biology, University of...collection E. coli DH5 Cloning host Laboratory collection BL21(DE3) Overproduction host Novagen GM1684 dam; for transformation of B. anthracis T. Koehler

  19. The molecular timeline of a reviving bacterial spore.

    Science.gov (United States)

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

    2015-02-19

    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.

  20. Sterilization of hydrogen peroxide resistant bacterial spores with stabilized chlorine dioxide.

    Science.gov (United States)

    Friedline, Anthony; Zachariah, Malcolm; Middaugh, Amy; Heiser, Matt; Khanna, Neeraj; Vaishampayan, Parag; Rice, Charles V

    2015-01-01

    Bacillus pumilus SAFR-032 spores isolated from a clean room environment are known to exhibit enhanced resistance to peroxide, desiccation, UV radiation and chemical disinfection than other spore-forming bacteria. The survival of B. pumilus SAFR-032 spores to standard clean room sterilization practices requires development of more stringent disinfection agents. Here, we report the effects of a stabilized chlorine dioxide-based biocidal agent against spores of B. pumilus SAFR-032 and Bacillus subtilis ATCC 6051. Viability was determined via CFU measurement after exposure. Chlorine dioxide demonstrated efficacy towards sterilization of spores of B. pumilus SAFR-032 equivalent or better than exposure to hydrogen peroxide. These results indicate efficacy of chlorine dioxide delivered through a stabilized chlorine dioxide product as a means of sterilization of peroxide- and UV-resistant spores.

  1. A simple identification method for spore-forming bacteria showing high resistance against [gamma]-rays

    Energy Technology Data Exchange (ETDEWEB)

    Koshikawa, Tomihiko; Sone, Koji; Kobayashi, Toshikazu (Japan Radioisotope Association, Koka, Shiga (Japan). Koka Lab.)

    1993-11-01

    A simple identification method was developed for spore-forming bacteria which are highly resistant against [gamma]-rays. Among 23 species of Bacillus studied, the spores of Bacillus megaterium, B. cereus, B. thuringiensis, B. pumilus and B. aneurinolyticus showed high resistance against [gamma]-rays as compared with other spores of Bacillus species. Combination of the seven kinds of biochemical tests, namely, the citrate utilization test, nitrate reduction test, starch hydrolysis test, Voges-Proskauer reaction test, gelatine hydrolysis test, mannitol utilization test and xylose utilization test showed a characteristic pattern for each species of Bacillus. The combination pattern of each the above tests with a few supplementary test, if necessary, was useful to identify Bacillus species showing high radiation resistance against [gamma]-rays. The method is specific for B. megaterium, B. thuringiensis and B. pumilus, and highly selective for B. aneurinolyticus and B. cereus. (author).

  2. The contribution of endogenous and exogenous effects to radiation-induced damage in the bacterial spore.

    Science.gov (United States)

    Jacobs, G P; Samuni, A; Czapski, G

    1985-06-01

    Radical scavengers such as polyethylene glycol 400 and 4000 and bovine albumin have been used to define the contribution of exogenous and endogenous effects to the gamma-radiation-induced damage in aqueous buffered suspensions of Bacillus pumilus spores. The results indicate that this damage in the bacterial spore is predominantly endogenous both in the presence of 1 atmosphere of oxygen, and in anoxia.

  3. Contribution of endogenous and exogenous effects to radiation-induced damage in the bacterial spore

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, G.P. (Hebrew Univ., Jerusalem (Israel). School of Pharmacy); Samuni, A. (Hebrew Univ., Jerusalem (Israel). School of Medicine); Czapski, G. (Hebrew Univ., Jerusalem (Israel). Dept. of Physical Chemistry)

    1985-06-01

    Radical scavengers such as polyethylene glycol 400 and 4000 and bovine albumin have been used to define the contribution of exogenous and endogenous effects to the gamma-radiation-induced damage in aqueous buffered suspensions of Bacillus pumilus spores. The results indicate that this damage in the bacterial spore is predominantly endogenous both in the presence of 1 atmosphere of oxygen, and in anoxia.

  4. Environmental microbiology as related to planetary quarantine. [water activity and temperature effects on bacterial spore survival

    Science.gov (United States)

    Pflug, I. J.

    1972-01-01

    The survival of Bacillus subtilis var. niger spores suspended in solutions of sucrose and glycerol at calculated water activities and varying temperatures was studied. The overall results indicated that as the water activity of the liquid decreased from .99 to .85, the heat resistance of the spores increased. The nature of the substance controlling the water activity, and the history of the spores prior to treatment also had an affect on their heat resistance.

  5. High resolution FESEM and TEM reveal bacterial spore attachment.

    Science.gov (United States)

    Panessa-Warren, Barbara J; Tortora, George T; Warren, John B

    2007-08-01

    Transmission electron microscopy (TEM) studies in the 1960s and early 1970s using conventional thin section and freeze fracture methodologies revealed ultrastructural bacterial spore appendages. However, the limited technology at that time necessitated the time-consuming process of imaging serial sections and reconstructing each structure. Consequently, the distribution and function of these appendages and their possible role in colonization or pathogenesis remained unknown. By combining high resolution field emission electron microscopy with TEM images of identical bacterial spore preparations, we have been able to obtain images of intact and sectioned Bacillus and Clostridial spores to clearly visualize the appearance, distribution, resistance (to trypsin, chloramphenicol, and heat), and participation of these structures to facilitate attachment of the spores to glass, agar, and human cell substrates. Current user-friendly commercial field emission scanning electron microscopes (FESEMs), permit high resolution imaging, with high brightness guns at lower accelerating voltages for beam sensitive intact biological samples, providing surface images at TEM magnifications for making direct comparisons. For the first time, attachment structures used by pathogenic, environmental, and thermophile bacterial spores could be readily visualized on intact spores to reveal how specific appendages and outer spore coats participated in spore attachment, colonization, and invasion.

  6. Improvement of immunodetection of bacterial spore antigen by ultrasonic cavitation.

    Science.gov (United States)

    Borthwick, Kathryn A J; Love, Tracey E; McDonnell, Martin B; Coakley, W Terence

    2005-11-15

    Ultrasonic cavitation was employed to enhance sensitivity of bacterial spore immunoassay detection, specifically, enzyme-linked immunosorbent assay (ELISA) and resonant mirror (RM) sensing. Bacillus spore suspensions were exposed to high-power ultrasound in a tubular sonicator operated at 267 kHz in both batch and flow modes. The sonicator was designed to deliver high output power and is in a form that can be cooled efficiently to avoid thermal denaturation of antigen. The 30-s batch and cooled flow (0.3 mL/min) sonication achieved an approximately 20-fold increase in ELISA sensitivity compared to unsonicated spores by ELISA. RM sensing of sonicated spores achieved detection sensitivity of approximately 10(6) spores/mL, whereas unsonicated spores were undetectable at the highest concentration tested. Improvements in detection were associated with antigen released from the spores. Equilibrium temperature increase in the tubular sonicator was limited to 14 K after 30 min and was maintained for 6 h with cooling and flow (0.3 mL/min). The work described here demonstrates the utility of the tubular sonicator for the improvement in the sensitivity of the detection of spores and its suitability as an in-line component of a rapid detection system.

  7. The Role of the Electrostatic Force in Spore Adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Eunhyea [Georgia Institute of Technology; Yiacoumi, Sotira [Georgia Institute of Technology; Lee, Ida [University of Tennessee, Knoxville (UTK); Tsouris, Costas [ORNL

    2010-01-01

    Electrostatic force is investigated as one of the components of the adhesion force between Bacillus thuringiensis (Bt) spores and planar surfaces. The surface potentials of a Bt spore and a mica surface are experimentally obtained using a combined atomic force microscopy (AFM)-scanning surface potential microscopy technique. On the basis of experimental information, the surface charge density of the spores is estimated at 0.03 {micro}C/cm{sup 2} at 20% relative humidity and decreases with increasing humidity. The Coulombic force is introduced for the spore-mica system (both charged, nonconductive surfaces), and an electrostatic image force is introduced to the spore-gold system because gold is electrically conductive. The Coulombic force for spore-mica is repulsive because the components are similarly charged, while the image force for the spore-gold system is attractive. The magnitude of both forces decreases with increasing humidity. The electrostatic forces are added to other force components, e.g., van der Waals and capillary forces, to obtain the adhesion force for each system. The adhesion forces measured by AFM are compared to the estimated values. It is shown that the electrostatic (Coulombic and image) forces play a significant role in the adhesion force between spores and planar surfaces.

  8. Sterilization Resistance of Bacterial Spores Explained with Water Chemistry.

    Science.gov (United States)

    Friedline, Anthony W; Zachariah, Malcolm M; Middaugh, Amy N; Garimella, Ravindranath; Vaishampayan, Parag A; Rice, Charles V

    2015-11-01

    Bacterial spores can survive for long periods without nutrients and in harsh environmental conditions. This survival is influenced by the structure of the spore, the presence of protective compounds, and water retention. These compounds, and the physical state of water in particular, allow some species of bacterial spores to survive sterilization schemes with hydrogen peroxide and UV light. The chemical nature of the spore core and its water has been a subject of some contention and the chemical environment of the water impacts resistance paradigms. Either the spore has a glassy core, where water is immobilized along with other core components, or the core is gel-like with mobile water diffusion. These properties affect the movement of peroxide and radical species, and hence resistance. Deuterium solid-state NMR experiments are useful for examining the nature of the water inside the spore. Previous work in our lab with spores of Bacillus subtilis indicate that, for spores, the core water is in a more immobilized state than expected for the gel-like core theory, suggesting a glassy core environment. Here, we report deuterium solid-state NMR observations of the water within UV- and peroxide-resistant spores from Bacillus pumilus SAFR-032. Variable-temperature NMR experiments indicate no change in the line shape after heating to 50 °C, but an overall decrease in signal after heating to 100 °C. These results show glass-like core dynamics within B. pumilus SAFR-032 that may be the potential source of its known UV-resistance properties. The observed NMR traits can be attributed to the presence of an exosporium containing additional labile deuterons that can aid in the deactivation of sterilizing agents.

  9. Small acid soluble proteins for rapid spore identification.

    Energy Technology Data Exchange (ETDEWEB)

    Branda, Steven S.; Lane, Todd W.; VanderNoot, Victoria A.; Jokerst, Amanda S.

    2006-12-01

    This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescence detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.

  10. In vitro high-resolution structural dynamics of single germinating bacterial spores

    Energy Technology Data Exchange (ETDEWEB)

    Plomp, M; Leighton, T; Wheeler, K; Malkin, A

    2006-11-14

    Although significant progress has been achieved in understanding the genetic and biochemical bases of the spore germination process, the structural basis for breaking the dormant spore state remains poorly understood. We have used atomic force microscopy (AFM) to probe the high-resolution structural dynamics of single Bacillus atrophaeus spores germinating under native conditions. Here we show that AFM can reveal previously unrecognized germination-induced alterations in spore coat architecture and topology as well as the disassembly of outer spore coat rodlet structures. These results and previous studies in other microorganisms suggest that the spore coat rodlets are structurally similar to amyloid fibrils. AFM analysis of the nascent surface of the emerging germ cell revealed a porous network of peptidoglycan fibers. The results are consistent with a honeycomb model structure for synthetic peptidoglycan oligomers determined by nuclear magnetic resonance. AFM is a promising experimental tool for investigating the morphogenesis of spore germination and cell wall peptidoglycan structure.

  11. In vitro high-resolution structural dynamics of single germinating bacterial spores

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence Livermore National Laboratory

    2006-12-11

    Although significant progress has been achieved in understanding the genetic and biochemical bases of the spore germination process, the structural basis for breaking the dormant spore state remains poorly understood. We have used atomic force microscopy (AFM) to probe the high-resolution structural dynamics of single Bacillus atrophaeus spores germinating under native conditions. Here we show that AFM can reveal previously unrecognized germination-induced alterations in spore coat architecture and topology as well as the disassembly of outer spore coat rodlet structures. These results and previous studies in other microorganisms suggest that the spore coat rodlets are structurally similar to amyloid fibrils. AFM analysis of the nascent surface of the emerging germ cell revealed a porous network of peptidoglycan fibers. The results are consistent with a honeycomb model structure for synthetic peptidoglycan oligomers determined by nuclear magnetic resonance. AFM is a promising experimental tool for investigating the morphogenesis of spore germination and cell wall peptidoglycan structure.

  12. Cryopreservation of fern spores

    Science.gov (United States)

    Spore banks for ferns are analogous to seed banks for angiosperms and provide a promising ex situ conservation tool because large quantities of germplasm with high genetic variation can be conserved in a small space with low economic and technical costs. Ferns produce two types of spores with very ...

  13. A versatile nano display platform from bacterial spore coat proteins.

    Science.gov (United States)

    Wu, I-Lin; Narayan, Kedar; Castaing, Jean-Philippe; Tian, Fang; Subramaniam, Sriram; Ramamurthi, Kumaran S

    2015-04-09

    Dormant bacterial spores are encased in a thick protein shell, the 'coat', which contains ∼70 different proteins. The coat protects the spore from environmental insults, and is among the most durable static structures in biology. Owing to extensive cross-linking among coat proteins, this structure has been recalcitrant to detailed biochemical analysis, so molecular details of how it assembles are largely unknown. Here, we reconstitute the basement layer of the coat atop spherical membranes supported by silica beads to create artificial spore-like particles. We report that these synthetic spore husk-encased lipid bilayers (SSHELs) assemble and polymerize into a static structure, mimicking in vivo basement layer assembly during sporulation in Bacillus subtilis. In addition, we demonstrate that SSHELs may be easily covalently modified with small molecules and proteins. We propose that SSHELs may be versatile display platforms for drugs and vaccines in clinical settings, or for enzymes that neutralize pollutants for environmental remediation.

  14. Spore-forming bacteria and their utilisation as probiotics.

    Science.gov (United States)

    Bader, J; Albin, A; Stahl, U

    2012-03-01

    In this review article, the beneficial application of bacterial spore formers as probiotics in the food industry is discussed based on the knowledge gleaned from current publications. The summary of new scientific results provides evidence of the advantages of the utilisation of Bacillus or Clostridium strains in the food industry. Both bacteria are able to produce a very stable duration form: the endospore. Compared to the widely used lactic acid bacteria, bacterial spores offer the advantage of a higher survival rate during the acidic stomach passage and better stability during the processing and storage of the food product. In many food products, germination of the spores does not occur. Hence the product quality of the food is not affected because of their inactive metabolism. Besides the possible utilisation and functional properties, an overview of the fast-developing knowledge about the mechanisms of the beneficial health effects of spore-forming bacteria is provided.

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

    Science.gov (United States)

    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

  16. Utility of sodium hypochlorite for ultrastructure study of bacterial spore integuments.

    Science.gov (United States)

    Rode, L J; Williams, M G

    1966-12-01

    Rode, L. J. (The University of Texas, Austin), and M. Glenn Williams. Utility of sodium hypochlorite for ultrastructure study of bacterial spore integuments. J. Bacteriol. 92:1772-1778. 1966.-Spores of Bacillus megaterium are partially dissolved by sodium hypochlorite. Spore integuments become visible during the dissolution, and ultrastructural features may be detected. Three distinct integument types are described for B. megaterium QM B1551 with the use of this technique. Since a variety of microbial cells are affected by sodium hypochlorite, its use may be applicable to ultrastructure study of cells other than bacterial spores.

  17. Sporulation of Bacillus spp. within biofilms: a potential source of contamination in food processing environments.

    Science.gov (United States)

    Faille, C; Bénézech, T; Midelet-Bourdin, G; Lequette, Y; Clarisse, M; Ronse, G; Ronse, A; Slomianny, C

    2014-06-01

    Bacillus strains are often isolated from biofilms in the food industries. Previous works have demonstrated that sporulation could occur in biofilms, suggesting that biofilms would be a significant source of food contamination with spores. In this study, we investigated the properties of mono-species and mixed Bacillus biofilms and the ability of Bacillus strains to sporulate inside biofilms. Bacillus strains were able to form mono-species biofilms on stainless steel coupons, with up to 90% spores after a 48 h-incubation. These spores were highly resistant to cleaning but were easily transferred to agar, mimicking the cross-contamination of food, thereby suggesting that biofilms would be of particular concern due to a potential for Bacillus spore food contamination. This hypothesis was strengthened by the fact that Bacillus strains were able to form mixed biofilms with resident strains and that sporulation still occurred easily in these complex structures.

  18. Fighting Ebola with novel spore decontamination technologies for the military

    Science.gov (United States)

    Doona, Christopher J.; Feeherry, Florence E.; Kustin, Kenneth; Olinger, Gene G.; Setlow, Peter; Malkin, Alexander J.; Leighton, Terrance

    2015-01-01

    Recently, global public health organizations such as Doctors without Borders (MSF), the World Health Organization (WHO), Public Health Canada, National Institutes of Health (NIH), and the U.S. government developed and deployed Field Decontamination Kits (FDKs), a novel, lightweight, compact, reusable decontamination technology to sterilize Ebola-contaminated medical devices at remote clinical sites lacking infra-structure in crisis-stricken regions of West Africa (medical waste materials are placed in bags and burned). The basis for effectuating sterilization with FDKs is chlorine dioxide (ClO2) produced from a patented invention developed by researchers at the US Army Natick Soldier RD&E Center (NSRDEC) and commercialized as a dry mixed-chemical for bacterial spore decontamination. In fact, the NSRDEC research scientists developed an ensemble of ClO2 technologies designed for different applications in decontaminating fresh produce; food contact and handling surfaces; personal protective equipment; textiles used in clothing, uniforms, tents, and shelters; graywater recycling; airplanes; surgical instruments; and hard surfaces in latrines, laundries, and deployable medical facilities. These examples demonstrate the far-reaching impact, adaptability, and versatility of these innovative technologies. We present herein the unique attributes of NSRDEC’s novel decontamination technologies and a Case Study of the development of FDKs that were deployed in West Africa by international public health organizations to sterilize Ebola-contaminated medical equipment. FDKs use bacterial spores as indicators of sterility. We review the properties and structures of spores and the mechanisms of bacterial spore inactivation by ClO2. We also review mechanisms of bacterial spore inactivation by novel, emerging, and established non-thermal technologies for food preservation, such as high pressure processing, irradiation, cold plasma, and chemical sanitizers, using an array of Bacillus

  19. Fighting Ebola with novel spore decontamination technologies for the military.

    Science.gov (United States)

    Doona, Christopher J; Feeherry, Florence E; Kustin, Kenneth; Olinger, Gene G; Setlow, Peter; Malkin, Alexander J; Leighton, Terrance

    2015-01-01

    Recently, global public health organizations such as Doctors without Borders (MSF), the World Health Organization (WHO), Public Health Canada, National Institutes of Health (NIH), and the U.S. government developed and deployed Field Decontamination Kits (FDKs), a novel, lightweight, compact, reusable decontamination technology to sterilize Ebola-contaminated medical devices at remote clinical sites lacking infra-structure in crisis-stricken regions of West Africa (medical waste materials are placed in bags and burned). The basis for effectuating sterilization with FDKs is chlorine dioxide (ClO2) produced from a patented invention developed by researchers at the US Army Natick Soldier RD&E Center (NSRDEC) and commercialized as a dry mixed-chemical for bacterial spore decontamination. In fact, the NSRDEC research scientists developed an ensemble of ClO2 technologies designed for different applications in decontaminating fresh produce; food contact and handling surfaces; personal protective equipment; textiles used in clothing, uniforms, tents, and shelters; graywater recycling; airplanes; surgical instruments; and hard surfaces in latrines, laundries, and deployable medical facilities. These examples demonstrate the far-reaching impact, adaptability, and versatility of these innovative technologies. We present herein the unique attributes of NSRDEC's novel decontamination technologies and a Case Study of the development of FDKs that were deployed in West Africa by international public health organizations to sterilize Ebola-contaminated medical equipment. FDKs use bacterial spores as indicators of sterility. We review the properties and structures of spores and the mechanisms of bacterial spore inactivation by ClO2. We also review mechanisms of bacterial spore inactivation by novel, emerging, and established non-thermal technologies for food preservation, such as high pressure processing, irradiation, cold plasma, and chemical sanitizers, using an array of Bacillus

  20. Scanning Surface Potential Microscopy of Spore Adhesion on Surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ida [University of Tennessee, Knoxville (UTK); Chung, Eunhyea [Georgia Institute of Technology; Kweon, Hyojin [Georgia Institute of Technology; Yiacoumi, Sotira [Georgia Institute of Technology; Tsouris, Costas [ORNL

    2012-01-01

    The adhesion of spores of Bacillus anthracis - the cause of anthrax and a likely biological threat - to solid surfaces is an important consideration in cleanup after an accidental or deliberate release. However, because of safety concerns, directly studying B. anthracis spores with advanced instrumentation is problematic. As a first step, we are examining the electrostatic potential of Bacillus thuringiensis (Bt), which is a closely related species that is often used as a simulant to study B. anthracis. Scanning surface potential microscopy (SSPM), also known as Kelvin probe force microscopy (KPFM), was used to investigate the influence of relative humidity (RH) on the surface electrostatic potential of Bt that had adhered to silica, mica, or gold substrates. AFM/SSPM side-by-side images were obtained separately in air, at various values of RH, after an aqueous droplet with spores was applied on each surface and allowed to dry before measurements. In the SSPM images, a negative potential on the surface of the spores was observed compared with that of the substrates. The surface potential decreased as the humidity increased. Spores were unable to adhere to a surface with an extremely negative potential, such as mica.

  1. Contribution of endogenous and exogenous damage to the total radiation-induced damage in the bacterial spore

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, G.P.; Samuni, A.; Czapski, G.

    1980-01-01

    Radical scavengers such as polyethylene glycol 4000 and bovine albumin have been used to define the contribution of exogenous and endogenous damage to the total radiation-induced damage in aqueous buffered suspensions of Bacillus pumilus spores. The results indicate that this damage in the bacterial spore is predominantly endogenous.

  2. Persistence of non-native spore forming bacteria in drinking water biofilm and evaluation of decontamination methods.

    Science.gov (United States)

    Shane, William T; Szabo, Jeffrey G; Bishop, Paul L

    2011-01-01

    Persistence of Bacillus globigii spores, a surrogate for Bacillus anthracis, was studied on biofouled concrete-lined slides in drinking water using biofilm annular reactors. Reactors were inoculated with B. globigii spores and persistence was monitored in the bulk and biofilm phases, first in dechlorinated water and later with free chlorine concentrations of 1 and 5 mg/L. In the dechlorinated study, a steady state population of spores developed on the slides. The addition of free chlorine at 5 mg/L decreased the adhered spore density by 2-logs within 4 hours and spores were not detected after 67 and 49 hours in the presence of 1 and 5 mg/L free chlorine, respectively. This suggests that adhered spores can persist in non-chlorinated conditions, but detach and/or are inactivated upon addition of free chlorine. When injected into a chlorinated reactor, adhered spore density continually decreased and spores were either undetectable or unquantifiable by 48 hours for both 1 and 5 mg/L chlorine concentrations. Results from these experiments suggest that the presence of a free chlorine residual limits adherence of viable spores to biofouled concrete-lined pipe walls by inactivating spores before they have attached. Both free chlorine concentrations (1 and 5 mg/L) were equally effective at inactivating spores in terms of log reduction, but the higher concentrations yielded faster rates of log reduction.

  3. Bacterial spores as particulate carriers for gene gun delivery of plasmid DNA.

    Science.gov (United States)

    Aps, Luana R M M; Tavares, Milene B; Rozenfeld, Julio H K; Lamy, M Teresa; Ferreira, Luís C S; Diniz, Mariana O

    2016-06-20

    Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles. More importantly, we demonstrated that a DNA vaccine adsorbed on spores can be loaded into biolistic cartridges and efficiently delivered into mice, which induced specific cellular and antibody responses. Altogether, these data indicate that B. subtilis spores represent a simple and low cost alternative for the in vivo delivery of DNA vaccines by the gene gun technology.

  4. Preparation and evaluation of spore-specific affinity- augmented bio-imprinted beads

    Energy Technology Data Exchange (ETDEWEB)

    Harvey, Scott D.; Mong, Gary M.; Ozanich, Rich M.; Mclean, Jeffrey S.; Goodwin, Shannon M.; Valentine, Nancy B.; Fredrickson, Jim K.

    2006-09-01

    The procedures previously described for imprinting bead surfaces with bacteria were applied to create novel affinity-augmented bacterial spore-imprinted beads. The imprinted beads are intended as a front-end spore capture/concentration stage of an integrated biological detection system. Our approach involves embedding bead surfaces with Bacillus thuringiensis kurstaki (Bt) spores (as a surrogate for Bacillus anthracis) during synthesis. Subsequent steps involved lithographic deactivation using a perfluoroether, spore removal to create imprint sites, and coating imprints with the lectin, concanavalin A, to provide general affinity. The synthesis of the intended material with the desired imprints was verified by scanning electron and confocal laser-scanning microscopy. The material was evaluated using spore-binding assays with either Bt or Bacillus subtilis (Bs) spores. The binding assays indicated strong spore-binding capability and a robust imprinting effect that accounted for 25 percent additional binding over nonimprinted controls. The binding assay results also indicated that further refinement of the surface deactivation procedure would enhance the performance of the imprinted substrate.

  5. Role of visible light-activated photocatalyst on the reduction of anthrax spore-induced mortality in mice.

    Directory of Open Access Journals (Sweden)

    Jyh-Hwa Kau

    Full Text Available BACKGROUND: Photocatalysis of titanium dioxide (TiO(2 substrates is primarily induced by ultraviolet light irradiation. Anion-doped TiO(2 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.

  6. Narrow terahertz attenuation signatures in Bacillus thuringiensis.

    Science.gov (United States)

    Zhang, Weidong; Brown, Elliott R; Viveros, Leamon; Burris, Kellie P; Stewart, C Neal

    2014-10-01

    Terahertz absorption signatures from culture-cultivated Bacillus thuringiensis were measured with a THz photomixing spectrometer operating from 400 to 1200 GHz. We observe two distinct signatures centered at ∼955 and 1015 GHz, and attribute them to the optically coupled particle vibrational resonance (surface phonon-polariton) of Bacillus spores. This demonstrates the potential of the THz attenuation signatures as "fingerprints" for label-free biomolecular detection.

  7. Bacillus thuringiensis as a surrogate for Bacillus anthracis in aerosol research.

    Science.gov (United States)

    Tufts, Jenia A M; Calfee, M Worth; Lee, Sang Don; Ryan, Shawn P

    2014-05-01

    Characterization of candidate surrogate spores prior to experimental use is critical to confirm that the surrogate characteristics are as closely similar as possible to those of the pathogenic agent of interest. This review compares the physical properties inherent to spores of Bacillus anthracis (Ba) and Bacillus thuringiensis (Bt) that impact their movement in air and interaction with surfaces, including size, shape, density, surface morphology, structure and hydrophobicity. Also evaluated is the impact of irradiation on the physical properties of both Bacillus species. Many physical features of Bt and Ba have been found to be similar and, while Bt is considered typically non-pathogenic, it is in the B. cereus group, as is Ba. When cultured and sporulated under similar conditions, both microorganisms share a similar cylindrical pellet shape, an aerodynamic diameter of approximately 1 μm (in the respirable size range), have an exosporium with a hairy nap, and have higher relative hydrophobicities than other Bacillus species. While spore size, morphology, and other physical properties can vary among strains of the same species, the variations can be due to growth/sporulation conditions and may, therefore, be controlled. Growth and sporulation conditions are likely among the most important factors that influence the representativeness of one species, or preparation, to another. All Bt spores may, therefore, not be representative of all Ba spores. Irradiated spores do not appear to be a good surrogate to predict the behavior of non-irradiated spores due to structural damage caused by the irradiation. While the use of Bt as a surrogate for Ba in aerosol testing appears to be well supported, this review does not attempt to narrow selection between Bt strains. Comparative studies should be performed to test the hypothesis that viable Ba and Bt spores will behave similarly when suspended in the air (as an aerosol) and to compare the known microscale characteristics

  8. Characterization of bacterial spore germination using integrated phase contrast microscopy, Raman spectroscopy, and optical tweezers.

    Science.gov (United States)

    Kong, Lingbo; Zhang, Pengfei; Setlow, Peter; Li, Yong-Qing

    2010-05-01

    We present a methodology that combines external phase contrast microscopy, Raman spectroscopy, and optical tweezers to monitor a variety of changes during the germination of single Bacillus cereus spores in both nutrient (l-alanine) and non-nutrient (Ca-dipicolinic acid (DPA)) germinants with a temporal resolution of approximately 2 s. Phase contrast microscopy assesses changes in refractility of individual spores during germination, while Raman spectroscopy gives information on changes in spore-specific molecules. The results obtained include (1) the brightness of the phase contrast image of an individual dormant spore is proportional to the level of CaDPA in that spore; (2) the end of the first Stage of germination, revealed as the end of the rapid drop in spore refractility by phase contrast microscopy, precisely corresponds to the completion of the release of CaDPA as revealed by Raman spectroscopy; and (3) the correspondence between the rapid drop in spore refractility and complete CaDPA release was observed not only for spores germinating in the well-controlled environment of an optical trap but also for spores germinating when adhered on a microscope coverslip. Using this latter method, we also simultaneously characterized the distribution of the time-to-complete-CaDPA release (T(release)) of hundreds of individual B. cereus spores germinating with both saturating and subsaturating concentrations of l-alanine and with CaDPA.

  9. 同时检测沙门氏菌和炭疽杆菌磁致伸缩生物传感器制备与应用%Preparation and application of magnetoelastic biosensors system for simultaneously detectingSalmonella typhimurium andBacillus anthracis spores

    Institute of Scientific and Technical Information of China (English)

    胡静; 胡佳佳; 沈雯; Bryan A Chin

    2016-01-01

    a biorecognition element. In this study, a magnetostrictive platform is served as the transducer, and as the mass sensitivity, the magnetoelastic resonance sensors have a characteristic resonant frequency that can be determined by monitoring the magnetic flux emitted by the sensor in response to an applied time-varying magnetic field. Due to the magnetoelastic nature of the amorphous magnetostrictive alloy, the sensor exhibits a physical resonance when it undergoes a time-varying magnetic field, and a shift in resonance frequency of the magnetostrictive sensor depends only on the mass change when environmental parameters are invariable. This magnetostrictive platform has a unique advantage over conventional sensor platforms in that its measurement is wireless and remote. And phage, which has been verified to be thermally stable, is used as the biorecognition element. In this paper, a multiple phage-based magnetoelastic (ME) biosenor system for simultaneously detectingSalmonella typhimuriumandBacillus anthracis spores was prepared by immobilizing 2 different kinds of phages as biorecognition element onto the magnetoelastic thin film made from 2826 MB MetglasTM, and the 2 kinds of phages were the E2 phage specific toSalmonella typhimurium and the JRB7 phage specific toBacillus anthracisspores, respectively. Finally, 1 mg/mL bovine serum albumin (BSA) was immobilized onto the magnetoelastic thin film as blocking agent for getting specific binding of target bacteria. The multiple phage-based magnetoelastic (ME) biosensor system was simultaneously monitored for the detection of different biological pathogens that were sequentially introduced to the measurement system.The detection system included a reference sensor as a control, an E2 phage-coated sensor specific toSalmonella typhimurium, and a JRB7 phage-coated sensor specific toBacillus anthracisspores. The sensors were free standing during the test, and held in place by a magnetic field. In the detection process, the

  10. Selective detection of 1000 B. anthracis spores within 15 minutes using a peptide functionalized SERS assay.

    Science.gov (United States)

    Farquharson, Stuart; Shende, Chetan; Smith, Wayne; Huang, Hermes; Inscore, Frank; Sengupta, Atanu; Sperry, Jay; Sickler, Todd; Prugh, Amber; Guicheteau, Jason

    2014-12-21

    A surface-enhanced Raman spectroscopy (SERS) assay has been designed to detect Bacillus anthracis spores. The assay consists of silver nanoparticles embedded in a porous glass structure that have been functionalized with ATYPLPIR, a peptide developed to discriminately bind B. anthracis versus other species of Bacillus. Once bound, acetic acid was used to release the biomarker dipicolinic acid from the spores, which was detected by SERS through the addition of silver colloids. This SERS assay was used to selectively bind B. anthracis with a 100-fold selectivity versus B. cereus, and to detect B. anthracis Ames at concentrations of 1000 spores per mL within 15 minutes. The SERS assay measurements provide a basis for the development of systems that can detect spores collected from the air or from water supplies.

  11. Activate to eradicate: inhibition of Clostridium difficile spore outgrowth by the synergistic effects of osmotic activation and nisin.

    Directory of Open Access Journals (Sweden)

    Michelle M Nerandzic

    Full Text Available BACKGROUND: Germination is the irreversible loss of spore-specific properties prior to outgrowth. Because germinating spores become more susceptible to killing by stressors, induction of germination has been proposed as a spore control strategy. However, this strategy is limited by superdormant spores that remain unaffected by germinants. Harsh chemicals and heat activation are effective for stimulating germination of superdormant spores but are impractical for use in a hospital setting, where Clostridium difficile spores present a challenge. Here, we tested whether osmotic activation solutes will provide a mild alternative for stimulation of superdormant C. difficile spores in the presence of germinants as previously demonstrated in several species of Bacillus. In addition, we tested the hypothesis that the limitations of superdormancy can be circumvented with a combined approach using nisin, a FDA-approved safe bacteriocin, to inhibit outgrowth of germinated spores and osmotic activation solutes to enhance outgrowth inhibition by stimulating superdormant spores. PRINCIPAL FINDINGS: Exposure to germination solution triggered ~1 log(10 colony forming units (CFU of spores to germinate, and heat activation increased the spores that germinated to >2.5 log(10CFU. Germinating spores, in contrast to dormant spores, became susceptible to inhibition by nisin. The presence of osmotic activation solutes did not stimulate germination of superdormant C. difficile spores exposed to germination solution. But, in the absence of germination solution, osmotic activation solutes enhanced nisin inhibition of superdormant spores to >3.5 log(10CFU. The synergistic effects of osmotic activation solutes and nisin were associated with loss of membrane integrity. CONCLUSIONS: These findings suggest that the synergistic effects of osmotic activation and nisin bypass the limitations of germination as a spore control strategy, and might be a novel method to safely and

  12. Pengaruh pH dan Perubahan Temperatur Terhadap Pembentukan Spora Bacillus sp. BK17

    OpenAIRE

    2014-01-01

    Bacterial spores are the surviving structure under unfavourable physical and chemical conditions. Bacillus sp. BK17 is a spore forming bacteria that has been reported to have an ability to inhibit the growth of various pathogenic fungi.This study aims to determine the best pH and temperature for the formation of spore. The result showed that Bacillus sp. BK17 has the highest spore formation at the initial pH of media of 5,0 and at a heat shock of 70° C for 60 minutes. 090805025

  13. New pressure and temperature effects on bacterial spores

    Energy Technology Data Exchange (ETDEWEB)

    Mathys, A; Knorr, D [Berlin University of Technology, Department of Food Biotechnology and Food Process Engineering, Koenigin-Luise-Str. 22, D-14195 Berlin (Germany); Heinz, V [German Institute of Food Technology, p. o. box 1165, D-49601, Quackenbrueck (Germany)], E-mail: alexander.mathys@tu-berlin.de

    2008-07-15

    The mechanism of inactivation of bacterial spores by heat and pressure is still a matter of discussion. Obviously, the change of the dissociation equilibrium under pressure and temperature plays a dominant role in inactivation of microorganisms. Heat and pressure inactivation of Geobacillus. stearothermophilus spores at different initial pH-values in ACES and phosphate buffer confirmed this view. Thermal inactivation in ACES buffer at 122 deg. C resulted in higher logarithmic reductions. Contrary, after pressure treatment at 900 MPa with 80 deg. C phosphate buffer showed higher inactivation. These results indicated the different dissociation equilibrium shifts in buffer systems by heat and pressure. Due to preparation, storage and handling of highly concentrated spore suspensions the clumping and the formation of aggregates can hardly be avoided. Consequently, the impact of the agglomeration size distribution on the quantitative assessment of G. stearothermophilus spore inactivation was determined by using a three-fold dynamic optical backreflexion measurement. Two limiting cases have been discriminated in mathematical modelling: three dimensional, spherical packing for maximum spore count and two dimensional, circular packing for minimum spore count of a particular agglomerate. Thermal inactivation studies have been carried out in thin glass capillaries, where by using numerical simulations the non isothermal conditions were modelled and taken into account. It is shown that the shoulder formation often found in thermal spore inactivation can sufficiently be described by first-order inactivation kinetics when the agglomeration size is considered. In case of high pressure inactivation agglomerations could be strongly changed by high forces at compression and especially decompression phase. The physiological response of Bacillus licheniformis spores to high pressure was investigated using multiparameter flow cytometry. Spores were treated by high pressure at 150 MPa

  14. New pressure and temperature effects on bacterial spores

    Science.gov (United States)

    Mathys, A.; Heinz, V.; Knorr, D.

    2008-07-01

    The mechanism of inactivation of bacterial spores by heat and pressure is still a matter of discussion. Obviously, the change of the dissociation equilibrium under pressure and temperature plays a dominant role in inactivation of microorganisms. Heat and pressure inactivation of Geobacillus. stearothermophilus spores at different initial pH-values in ACES and phosphate buffer confirmed this view. Thermal inactivation in ACES buffer at 122°C resulted in higher logarithmic reductions. Contrary, after pressure treatment at 900 MPa with 80°C phosphate buffer showed higher inactivation. These results indicated the different dissociation equilibrium shifts in buffer systems by heat and pressure. Due to preparation, storage and handling of highly concentrated spore suspensions the clumping and the formation of aggregates can hardly be avoided. Consequently, the impact of the agglomeration size distribution on the quantitative assessment of G. stearothermophilus spore inactivation was determined by using a three-fold dynamic optical backreflexion measurement. Two limiting cases have been discriminated in mathematical modelling: three dimensional, spherical packing for maximum spore count and two dimensional, circular packing for minimum spore count of a particular agglomerate. Thermal inactivation studies have been carried out in thin glass capillaries, where by using numerical simulations the non isothermal conditions were modelled and taken into account. It is shown that the shoulder formation often found in thermal spore inactivation can sufficiently be described by first-order inactivation kinetics when the agglomeration size is considered. In case of high pressure inactivation agglomerations could be strongly changed by high forces at compression and especially decompression phase. The physiological response of Bacillus licheniformis spores to high pressure was investigated using multiparameter flow cytometry. Spores were treated by high pressure at 150 MPa with 37

  15. Ribosomal ribonucleic acid maturation during bacterial spore germination.

    Science.gov (United States)

    Bleyman, M; Woese, C

    1969-01-01

    All the ribosomal ribonucleic acid made during the early stages of germination of spores of Bacillus subtilis is of the "precursor" type, i.e., that type appearing in the incomplete forms of the ribosome. Shortly before the onset of deoxyribonucleic acid synthesis in germination, this precursor ribonucleic acid changed to the mature ribosomal ribonucleic acid characteristic of the 30S and 50S ribosomal subunits.

  16. Kinetics of germination of individual spores of Geobacillus stearothermophilus as measured by raman spectroscopy and differential interference contrast microscopy.

    Directory of Open Access Journals (Sweden)

    Tingting Zhou

    Full Text Available 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 DT(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 DT(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 T(lag 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.

  17. Fifth international fungus spore conference

    Energy Technology Data Exchange (ETDEWEB)

    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.

  18. The use of bacterial spore formers as probiotics.

    Science.gov (United States)

    Hong, Huynh A; Duc, Le Hong; Cutting, Simon M

    2005-09-01

    The field of probiosis has emerged as a new science with applications in farming and aqaculture as alternatives to antibiotics as well as prophylactics in humans. Probiotics are being developed commercially for both human use, primarily as novel foods or dietary supplements, and in animal feeds for the prevention of gastrointestinal infections, with extensive use in the poultry and aquaculture industries. The impending ban of antibiotics in animal feed, the current concern over the spread of antibiotic resistance genes, the failure to identify new antibiotics and the inherent problems with developing new vaccines make a compelling case for developing alternative prophylactics. Among the large number of probiotic products in use today are bacterial spore formers, mostly of the genus Bacillus. Used primarily in their spore form, these products have been shown to prevent gastrointestinal disorders and the diversity of species used and their applications are astonishing. Understanding the nature of this probiotic effect is complicated, not only because of the complexities of understanding the microbial interactions that occur within the gastrointestinal tract (GIT), but also because Bacillus species are considered allochthonous microorganisms. This review summarizes the commercial applications of Bacillus probiotics. A case will be made that many Bacillus species should not be considered allochthonous microorganisms but, instead, ones that have a bimodal life cycle of growth and sporulation in the environment as well as within the GIT. Specific mechanisms for how Bacillus species can inhibit gastrointestinal infections will be covered, including immunomodulation and the synthesis of antimicrobials. Finally, the safety and licensing issues that affect the use of Bacillus species for commercial development will be summarized, together with evidence showing the growing need to evaluate the safety of individual Bacillus strains as well as species on a case by case by basis.

  19. Potential role of Bacillus endospores in soil amended by olive mill wastewater.

    Science.gov (United States)

    Naclerio, Gino; Falasca, Antonio; Petrella, Emma; Nerone, Valentina; Cocco, Federica; Celico, Fulvio

    2010-01-01

    The main aim of this work was to know how spread is laccase activity in spores of Bacillus species isolated from a soil where Italian law allows olive mill wastewater (OMW) spreading, and to investigate the potential role of such autochthonous soil microorganisms in degradation of OMW phenols, and prevention of groundwater pollution. Laccase activity was detected for the first time in spores of wild-type Bacillus pumilus, B. cereus sensu lato, and B. amyloliquefaciens strains. Because B. pumilus, B. cereus sensu lato, and B. amyloliquefaciens, together with B. subtilis account for a total of 93% of Bacillus isolates at the study site, the nearly totality of Bacillus spores reveals laccase activity. Thus, taking also into consideration that Bacillus spores are more abundant (about 100-fold) than white-rot fungi (that possess a well known extracellular, radical-based ligninolytic enzyme system capable of degrading OMW phenols) in the studied soil, these spores may contribute to in-situ degradation of OMW phenols. This role is further emphasized by dilution of crude OMW during infiltration of rainwater through soil that allows to minimize the antibacterial activity of phenols. The widespread presence of Bacillus spores in soils indicates a potential detoxifying role of these spores in a broader context.

  20. Protection of bacterial spores in space, a contribution to the discussion on Panspermia.

    Science.gov (United States)

    Horneck, G; Rettberg, P; Reitz, G; Wehner, J; Eschweiler, U; Strauch, K; Panitz, C; Starke, V; Baumstark-Khan, C

    2001-12-01

    Spores of Bacillus subtilis were exposed to space in the BIOPAN facility of the European Space Agency onboard of the Russian Earth-orbiting FOTON satellite. The spores were exposed either in dry layers without any protecting agent, or mixed with clay, red sandstone, Martian analogue soil or meteorite powder, in dry layers as well as in so-called 'artificial meteorites', i.e. cubes filled with clay and spores in naturally occurring concentrations. After about 2 weeks in space, their survival was tested from the number of colony formers. Unprotected spores in layers open to space or behind a quartz window were completely or nearly completely inactivated (survival rates in most cases Panspermia, may not provide sufficient protection for spores to survive. The data are also pertinent to search for life on Mars and planetary protection considerations for future missions to Mars.

  1. In pursuit of protein targets: proteomic characterization of bacterial spore outer layers.

    Science.gov (United States)

    Abhyankar, Wishwas; Hossain, Abeer H; Djajasaputra, André; Permpoonpattana, Patima; Ter Beek, Alexander; Dekker, Henk L; Cutting, Simon M; Brul, Stanley; de Koning, Leo J; de Koster, Chris G

    2013-10-04

    Bacillus cereus, responsible for food poisoning, and Clostridium difficile, the causative agent of Clostridium difficile-associated diarrhea (CDAD), are both spore-forming pathogens involved in food spoilage, food intoxication, and other infections in humans and animals. The proteinaceous coat and the exosporium layers from spores are important for their resistance and pathogenicity characteristics. The exosporium additionally provides an ability to adhere to surfaces eventually leading to spore survival in food. Thus, studying these layers and identifying suitable protein targets for rapid detection and removal of spores is of the utmost importance. In this study, we identified 100 proteins from B. cereus spore coat, exosporium and 54 proteins from the C. difficile coat insoluble protein fraction. In an attempt to define a universal set of spore outer layer proteins, we identified 11 superfamily domains common to the identified proteins from two Bacilli and one Clostridium species. The evaluated orthologue relationships of identified proteins across different spore formers resulted in a set of 13 coat proteins conserved across the spore formers and 12 exosporium proteins conserved in the B. cereus group, which could be tested for quick and easy detection or targeted in strategies aimed at removal of spores from surfaces.

  2. Bacillus coagulans

    Science.gov (United States)

    Bacillus coagulans is a type of bacteria. It is used similarly to lactobacillus and other probiotics as "beneficial" bacteria. People take Bacillus coagulans for diarrhea, including infectious types such as ...

  3. Spore: Spawning Evolutionary Misconceptions?

    Science.gov (United States)

    Bean, Thomas E.; Sinatra, Gale M.; Schrader, P. G.

    2010-10-01

    The use of computer simulations as educational tools may afford the means to develop understanding of evolution as a natural, emergent, and decentralized process. However, special consideration of developmental constraints on learning may be necessary when using these technologies. Specifically, the essentialist (biological forms possess an immutable essence), teleological (assignment of purpose to living things and/or parts of living things that may not be purposeful), and intentionality (assumption that events are caused by an intelligent agent) biases may be reinforced through the use of computer simulations, rather than addressed with instruction. We examine the video game Spore for its depiction of evolutionary content and its potential to reinforce these cognitive biases. In particular, we discuss three pedagogical strategies to mitigate weaknesses of Spore and other computer simulations: directly targeting misconceptions through refutational approaches, targeting specific principles of scientific inquiry, and directly addressing issues related to models as cognitive tools.

  4. New Rapid Spore Assay

    Science.gov (United States)

    Kminek, Gerhard; Conley, Catharine

    2012-07-01

    The presentation will detail approved Planetary Protection specifications for the Rapid Spore Assay for spacecraft components and subsystems. Outlined will be the research and studies on which the specifications were based. The research, funded by ESA and NASA/JPL, was conducted over a period of two years and was followed by limited cleanroom studies to assess the feasibility of this assay during spacecraft assembly.

  5. Spore coat architecture of Clostridium novyi NT spores.

    Science.gov (United States)

    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.

  6. Clostridium difficile spore-macrophage interactions: spore survival.

    Directory of Open Access Journals (Sweden)

    Daniel Paredes-Sabja

    Full Text Available BACKGROUND: Clostridium difficile is the main cause of nosocomial infections including antibiotic associated diarrhea, pseudomembranous colitis and toxic megacolon. During the course of Clostridium difficile infections (CDI, C. difficile undergoes sporulation and releases spores to the colonic environment. The elevated relapse rates of CDI suggest that C. difficile spores has a mechanism(s to efficiently persist in the host colonic environment. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we provide evidence that C. difficile spores are well suited to survive the host's innate immune system. Electron microscopy results show that C. difficile spores are recognized by discrete patchy regions on the surface of macrophage Raw 264.7 cells, and phagocytosis was actin polymerization dependent. Fluorescence microscopy results show that >80% of Raw 264.7 cells had at least one C. difficile spore adhered, and that ∼60% of C. difficile spores were phagocytosed by Raw 264.7 cells. Strikingly, presence of complement decreased Raw 264.7 cells' ability to phagocytose C. difficile spores. Due to the ability of C. difficile spores to remain dormant inside Raw 264.7 cells, they were able to survive up to 72 h of macrophage infection. Interestingly, transmission electron micrographs showed interactions between the surface proteins of C. difficile spores and the phagosome membrane of Raw 264.7 cells. In addition, infection of Raw 264.7 cells with C. difficile spores for 48 h produced significant Raw 264.7 cell death as demonstrated by trypan blue assay, and nuclei staining by ethidium homodimer-1. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that despite efficient recognition and phagocytosis of C. difficile spores by Raw 264.7 cells, spores remain dormant and are able to survive and produce cytotoxic effects on Raw 264.7 cells.

  7. Bacterial spore inhibition and inactivation in foods by pressure, chemical preservatives, and mild heat.

    Science.gov (United States)

    Shearer, A E; Dunne, C P; Sikes, A; Hoover, D G

    2000-11-01

    Sucrose laurates, sucrose palmitate, sucrose stearates, and monolaurin (Lauricidin) were evaluated for inhibitory effects against spores of Bacillus sp., Clostridium sporogenes PA3679, and Alicyclobacillus sp. in a model agar system. The combined treatment of sucrose laurate, high hydrostatic pressure, and mild heat was evaluated on spores of Bacillus and Alicyclobacillus in foods. The minimum inhibitory concentrations of the sucrose esters were higher than that of Lauricidin for all spores tested in the model agar system, but Lauricidin was not the most readily suspended in the test media. The sucrose laurates and sucrose palmitate were more effective and more readily suspended than the sucrose stearates. A combined treatment of sucrose laurate (<1.0%), 392 megaPascals (MPa) at 45 degrees C for 10 to 15 min provided 3- to 5.5-log10 CFU/ml reductions from initial populations of 10(6) CFU/ml for Bacillus subtilis 168 in milk, Bacillus cereus 14579 in beef, Bacillus coagulans 7050 in tomato juice (pH 4.5), Alicyclobacillus sp. N1089 in tomato juice (pH 4.5), and Alicyclobacillus sp. N1098 in apple juice. The most notable change in the appearance of the products was temporary foaming during mixing of the sucrose laurate in the foods. The effect of sucrose laurate appeared to be inhibitory rather than lethal to the spores. The inhibitory effects observed on Bacillus and Alicyclobacillus spores by the combined treatment of pressure, mild heat, and sucrose laurate appear promising for food applications where alternatives to high heat processing are desired.

  8. Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

    Energy Technology Data Exchange (ETDEWEB)

    Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I. (Department of Agriculture, College Station, TX (USA))

    1990-08-01

    Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.

  9. Dipicolinic Acid Release by Germinating Clostridium difficile Spores Occurs through a Mechanosensing Mechanism

    Science.gov (United States)

    Francis, Michael B.

    2016-01-01

    ABSTRACT 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

  10. Bacterial spore survival after exposure to HZE particle bombardment -implication for the lithopanspermia hypothesis.

    Science.gov (United States)

    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

  11. Crossing of the epithelial barriers by Bacillus anthracis: the Known and the Unknown

    OpenAIRE

    2015-01-01

    Anthrax, caused by Bacillus anthracis, a Gram-positive spore-forming bacterium, is initiated by the entry of spores into the host body. There are three types of human infection: cutaneous, inhalational, and gastrointestinal. For each form, B. anthracis spores need to cross the cutaneous, respiratory or digestive epithelial barriers, respectively, as a first obligate step to establish infection. Anthrax is a toxi-infection: an association of toxemia and rapidly spreading infection progressing ...

  12. Regulation of Growth of the Mother Cell and Chromosome Replication during Sporulation of Bacillus subtilis ▿

    OpenAIRE

    Xenopoulos, Panagiotis; Piggot, Patrick J.

    2011-01-01

    During spore formation, Bacillus subtilis divides asymmetrically, resulting in two cells with different fates. Immediately after division, the transcription factor σF becomes active in the smaller prespore, followed by activation of σE in the larger mother cell. We recently showed that a delay in σE activation resulted in the novel phenotype of two spores (twins) forming within the same mother cell. Mother cells bearing twins are substantially longer than mother cells with single spores. Here...

  13. In pursuit of protein targets: proteomic characterization of bacterial spore outer layers

    NARCIS (Netherlands)

    Abhyankar, W.; Hossain, A.H.; Djajasaputra, A.; Permpoonpattana, P.; ter Beek, A.; Dekker, H.L.; Cutting, S.M.; Brul, S.; de Koning, L.J.; de Koster, C.G.

    2013-01-01

    Bacillus cereus, responsible for food poisoning, and Clostridium difficile, the causative agent of Clostridium difficile-associated diarrhea (CDAD), are both spore-forming pathogens involved in food spoilage, food intoxication, and other infections in humans and animals. The proteinaceous coat and t

  14. High gas pressure: an innovative method for the inactivation of dried bacterial spores.

    Science.gov (United States)

    Colas de la Noue, A; Espinasse, V; Perrier-Cornet, J-M; Gervais, P

    2012-08-01

    In this article, an original non-thermal process to inactivate dehydrated bacterial spores is described. The use of gases such as nitrogen or argon as transmission media under high isostatic pressure led to an inactivation of over 2 logs CFU/g of Bacillus subtilis spores at 430 MPa, room temperature, for a 1 min treatment. A major requirement for the effectiveness of the process resided in the highly dehydrated state of the spores. Only a water activity below 0.3 led to substantial inactivation. The solubility of the gas in the lipid components of the spore and its diffusion properties was essential to inactivation. The main phenomenon involved seems to be the sorption of the gas under pressure by the spores' structures such as residual pores and plasma membranes, followed by a sudden drop in pressure. Observation by phase-contrast microscopy suggests that internal structures have been affected by the treatment. Some parallels with polymer permeability to gas and rigidity at various water activities offer a few clues about the behavior of the outer layers of spores in response to this parameter and provide a good explanation for the sensitivity of spores to high gas pressure discharge at low hydration levels. Specificity of microorganisms such as size, organization, and composition could help in understanding the differences between spores and yeast regarding the parameters required for inactivation, such as pressure or maintenance time.

  15. New insights in the bacterial spore resistance to extreme terrestrial and extraterrestrial factors

    Science.gov (United States)

    Moeller, Ralf; Horneck, Gerda; Reitz, Guenther

    Based on their unique resistance to various space parameters, Bacillus endospores are one of the model systems used for astrobiological studies. The extremely high resistance of bacterial endospores to environmental stress factors has intrigued researchers since long time and many characteristic spore features, especially those involved in the protection of spore DNA, have already been uncovered. The disclosure of the complete genomic sequence of Bacillus subtilis 168, one of the often used astrobiological model system, and the rapid development of tran-scriptional microarray techniques have opened new opportunities of gaining further insights in the enigma of spore resistance. Spores of B. subtilis were exposed to various extreme ter-restrial and extraterrestrial stressors to reach a better understanding of the DNA protection and repair strategies, which them to cope with the induced DNA damage. Following physical stress factors of environmental importance -either on Earth or in space -were selected for this thesis: (i) mono-and polychromatic UV radiation, (ii) ionizing radiation, (iii) exposure to ultrahigh vacuum; and (iv) high shock pressures simulating meteorite impacts. To reach a most comprehensive understanding of spore resistance to those harsh terrestrial or simulated extraterrestrial conditions, a standardized experimental protocol of the preparation and ana-lyzing methods was established including the determination of the following spore responses: (i) survival, (ii) induced mutations, (iii) DNA damage, (iv) role of different repair pathways by use of a set of repair deficient mutants, and (v) transcriptional responses during spore germi-nation by use of genome-wide transcriptome analyses and confirmation by RT-PCR. From this comprehensive set of data on spore resistance to a variety of environmental stress parameters a model of a "built-in" transcriptional program of bacterial spores in response to DNA damaging treatments to ensure DNA restoration

  16. Bacillus anthracis factors for phagosomal escape.

    Science.gov (United States)

    Tonello, Fiorella; Zornetta, Irene

    2012-07-01

    The mechanism of phagosome escape by intracellular pathogens is an important step in the infectious cycle. During the establishment of anthrax, Bacillus anthracis undergoes a transient intracellular phase in which spores are engulfed by local phagocytes. Spores germinate inside phagosomes and grow to vegetative bacilli, which emerge from their resident intracellular compartments, replicate and eventually exit from the plasma membrane. During germination, B. anthracis secretes multiple factors that can help its resistance to the phagocytes. Here the possible role of B. anthracis toxins, phospholipases, antioxidant enzymes and capsules in the phagosomal escape and survival, is analyzed and compared with that of factors of other microbial pathogens involved in the same type of process.

  17. Glycosylation of BclA Glycoprotein from Bacillus cereus and Bacillus anthracis Exosporium Is Domain-specific.

    Science.gov (United States)

    Maes, Emmanuel; Krzewinski, Frederic; Garenaux, Estelle; Lequette, Yannick; Coddeville, Bernadette; Trivelli, Xavier; Ronse, Annette; Faille, Christine; Guerardel, Yann

    2016-04-29

    The spores of the Bacillus cereus group (B. cereus, Bacillus anthracis, and Bacillus thuringiensis) are surrounded by a paracrystalline flexible yet resistant layer called exosporium that plays a major role in spore adhesion and virulence. The major constituent of its hairlike surface, the trimerized glycoprotein BclA, is attached to the basal layer through an N-terminal domain. It is then followed by a repetitive collagen-like neck bearing a globular head (C-terminal domain) that promotes glycoprotein trimerization. The collagen-like region of B. anthracis is known to be densely substituted by unusual O-glycans that may be used for developing species-specific diagnostics of B. anthracis spores and thus targeted therapeutic interventions. In the present study, we have explored the species and domain specificity of BclA glycosylation within the B. cereus group. First, we have established that the collagen-like regions of both B. anthracis and B. cereus are similarly substituted by short O-glycans that bear the species-specific deoxyhexose residues anthrose and the newly observed cereose, respectively. Second we have discovered that the C-terminal globular domains of BclA from both species are substituted by polysaccharide-like O-linked glycans whose structures are also species-specific. The presence of large carbohydrate polymers covering the surface of Bacillus spores may have a profound impact on the way that spores regulate their interactions with biotic and abiotic surfaces and represents potential new diagnostic targets.

  18. The Bacillus anthracis Exosporium: What's the Big "Hairy" Deal?

    Science.gov (United States)

    Bozue, Joel A; Welkos, Susan; Cote, Christopher K

    2015-10-01

    In some Bacillus species, including Bacillus subtilis, the coat is the outermost layer of the spore. In others, such as the Bacillus cereus family, there is an additional layer that envelops the coat, called the exosporium. In the case of Bacillus anthracis, a series of fine hair-like projections, also referred to as a "hairy" nap, extends from the exosporium basal layer. The exact role of the exosporium in B. anthracis, or for any of the Bacillus species possessing this structure, remains unclear. However, it has been assumed that the exosporium would play some role in infection for B. anthracis, because it is the outermost structure of the spore and would make initial contact with host and immune cells during infection. Therefore, the exosporium has been a topic of great interest, and over the past decade much progress has been made to understand its composition, biosynthesis, and potential roles. Several key aspects of this spore structure, however, are still debated and remain undetermined. Although insights have been gained on the interaction of exosporium with the host during infection, the exact role and significance of this complex structure remain to be determined. Furthermore, because the exosporium is a highly antigenic structure, future strategies for the next-generation anthrax vaccine should pursue its inclusion as a component to provide protection against the spore itself during the initial stages of anthrax.

  19. Refined multivalent display of bacterial spore-binding peptides.

    Science.gov (United States)

    Lusvarghi, Sabrina; Kim, Jenny Morana; Creeger, Yehuda; Armitage, Bruce Alan

    2009-05-07

    A multiple antigen peptide display scaffold was used to create multivalent versions of a heptapeptide selected previously by phage display to bind to Bacillus subtilis spores. A simple flow cytometric assay was developed in which a biotinylated form of the peptide was first bound to fluorescent streptavidin, then the fluorescent streptavidin-peptide complex was bound to spores before introduction into the cytometer. This assay clearly demonstrated that the tetravalent scaffold enhanced the affinity for B. subtilis spores by greater than 1 and 2 orders of magnitude when compared to divalent and monovalent analogues, respectively. However, variations in the number and flexibility of spacer residues within the scaffold did not significantly affect the binding affinity of the tetravalent peptides. Similar to prior reports, these multivalent scaffolds are effective most likely because they mimic the multivalent display of the original peptide library on the phage coat. Moreover, the tetravalent peptides can be readily integrated into a variety of heterogeneous and homogeneous spore-detection assay formats.

  20. Validation of a nylon-flocked-swab protocol for efficient recovery of bacterial spores from smooth and rough surfaces.

    Science.gov (United States)

    Probst, Alexander; Facius, Rainer; Wirth, Reinhard; Moissl-Eichinger, Christine

    2010-08-01

    In order to meet planetary-protection requirements, culturable bacterial spore loads are measured representatively for the total microbial contamination of spacecraft. However, the National Aeronautics and Space Administration's (NASA's) cotton swab protocols for spore load determination have not changed for decades. To determine whether a more efficient alternative was available, a novel swab was evaluated for recovery of different Bacillus atrophaeus spore concentrations on stainless steel and other surfaces. Two protocols for the nylon-flocked swab (NFS) were validated and compared to the present NASA standard protocol. The results indicate that the novel swab protocols recover 3- to 4-fold more (45.4% and 49.0% recovery efficiency) B. atrophaeus spores than the NASA standard method (13.2%). Moreover, the nylon-flocked-swab protocols were superior in recovery efficiency for spores of seven different Bacillus species, including Bacillus anthracis Sterne (recovery efficiency, 20%). The recovery efficiencies for B. atrophaeus spores from different surfaces showed a variation from 5.9 to 62.0%, depending on the roughness of the surface analyzed. Direct inoculation of the swab resulted in a recovery rate of about 80%, consistent with the results of scanning electron micrographs that allowed detailed comparisons of the two swab types. The results of this investigation will significantly contribute to the cleanliness control of future life detection missions and will provide significant improvement in detection of B. anthracis contamination for law enforcement and security efforts.

  1. Detection and differentiation of bacterial spores in a mineral matrix by Fourier transform infrared spectroscopy (FTIR and chemometrical data treatment

    Directory of Open Access Journals (Sweden)

    Brandes Ammann Andrea

    2011-07-01

    Full Text Available Abstract Background Fourier transform infrared spectroscopy (FTIR has been used as analytical tool in chemistry for many years. In addition, FTIR can also be applied as a rapid and non-invasive method to detect and identify microorganisms. The specific and fingerprint-like spectra allow - under optimal conditions - discrimination down to the species level. The aim of this study was to develop a fast and reproducible non-molecular method to differentiate pure samples of Bacillus spores originating from different species as well as to identify spores in a simple matrix, such as the clay mineral, bentonite. Results We investigated spores from pure cultures of seven different Bacillus species by FTIR in reflection or transmission mode followed by chemometrical data treatment. All species investigated (B. atrophaeus, B. brevis, B. circulans, B. lentus, B. megaterium, B. subtilis, B. thuringiensis are typical aerobic soil-borne spore formers. Additionally, a solid matrix (bentonite and mixtures of benonite with spores of B. megaterium at various wt/wt ratios were included in the study. Both hierarchical cluster analysis and principal component analysis of the spectra along with multidimensional scaling allowed the discrimination of different species and spore-matrix-mixtures. Conclusions Our results show that FTIR spectroscopy is a fast method for species-level discrimination of Bacillus spores. Spores were still detectable in the presence of the clay mineral bentonite. Even a tenfold excess of bentonite (corresponding to 2.1 × 1010 colony forming units per gram of mineral matrix still resulted in an unambiguous identification of B. megaterium spores.

  2. Semi-automated bacterial spore detection system with micro-fluidic chips for aerosol collection, spore treatment and ICAN DNA detection.

    Science.gov (United States)

    Inami, Hisao; Tsuge, Kouichiro; Matsuzawa, Mitsuhiro; Sasaki, Yasuhiko; Togashi, Shigenori; Komano, Asuka; Seto, Yasuo

    2009-07-15

    A semi-automated bacterial spore detection system (BSDS) was developed to detect biological threat agents (e.g., Bacillus anthracis) on-site. The system comprised an aerosol sampler, micro-fluidic chip-A (for spore germination and cell lysis), micro-fluidic chip-B (for extraction and detection of genomic DNA) and an analyzer. An aerosol with bacterial spores was first collected in the collection chamber of chip-A with a velocity of 300 l/min, and the chip-A was taken off from the aerosol sampler and loaded into the analyzer. Reagents packaged in the chip-A were sequentially applied into the chamber. The genomic DNA extract from spore lyzate was manually transferred from chip-A to chip-B and loaded into the analyzer. Genomic DNA in chip-B was first trapped on a glass bead column, washed with various reagents, and eluted to the detection chamber by sequential auto-dispensing. Isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) with fluorescent measurement was adopted to amplify and detect target DNA. Bacillus subtilis was the stimulant of biological warfare agent in this experiment. Pretreatment conditions were optimized by examining bacterial target DNA recovery in the respective steps (aerosol collection, spore germination, cell lysis, and DNA extraction), by an off-chip experiment using a real-time polymerase chain reaction quantification method. Without the germination step, B. subtilis spores did not demonstrate amplification of target DNA. The detection of 10(4) spores was achieved within 2h throughout the micro-fluidic process.

  3. Characterization of aerobic spore-forming bacteria associated with industrial dairy processing environments and product spoilage.

    Science.gov (United States)

    Lücking, Genia; Stoeckel, Marina; Atamer, Zeynep; Hinrichs, Jörg; Ehling-Schulz, Monika

    2013-09-02

    Due to changes in the design of industrial food processing and increasing international trade, highly thermoresistant spore-forming bacteria are an emerging problem in food production. Minimally processed foods and products with extended shelf life, such as milk products, are at special risk for contamination and subsequent product damages, but information about origin and food quality related properties of highly heat-resistant spore-formers is still limited. Therefore, the aim of this study was to determine the biodiversity, heat resistance, and food quality and safety affecting characteristics of aerobic spore-formers in the dairy sector. Thus, a comprehensive panel of strains (n=467), which originated from dairy processing environments, raw materials and processed foods, was compiled. The set included isolates associated with recent food spoilage cases and product damages as well as isolates not linked to product spoilage. Identification of the isolates by means of Fourier-transform infrared spectroscopy and molecular methods revealed a large biodiversity of spore-formers, especially among the spoilage associated isolates. These could be assigned to 43 species, representing 11 genera, with Bacillus cereus s.l. and Bacillus licheniformis being predominant. A screening for isolates forming thermoresistant spores (TRS, surviving 100°C, 20 min) showed that about one third of the tested spore-formers was heat-resistant, with Bacillus subtilis and Geobacillus stearothermophilus being the prevalent species. Strains producing highly thermoresistant spores (HTRS, surviving 125°C, 30 min) were found among mesophilic as well as among thermophilic species. B. subtilis and Bacillus amyloliquefaciens were dominating the group of mesophilic HTRS, while Bacillus smithii and Geobacillus pallidus were dominating the group of thermophilic HTRS. Analysis of spoilage-related enzymes of the TRS isolates showed that mesophilic strains, belonging to the B. subtilis and B. cereus

  4. Complete Genomes of Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, Two Phylogenetically Distinct Probiotics.

    Directory of Open Access Journals (Sweden)

    Indu Khatri

    Full Text Available Several spore-forming strains of Bacillus are marketed as probiotics due to their ability to survive harsh gastrointestinal conditions and confer health benefits to the host. We report the complete genomes of two commercially available probiotics, Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, and compare them with the genomes of other Bacillus and Lactobacillus. The taxonomic position of both organisms was established with a maximum-likelihood tree based on twenty six housekeeping proteins. Analysis of all probiotic strains of Bacillus and Lactobacillus reveal that the essential sporulation proteins are conserved in all Bacillus probiotic strains while they are absent in Lactobacillus spp. We identified various antibiotic resistance, stress-related, and adhesion-related domains in these organisms, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival during antibiotic treatment and harsh conditions.

  5. Complete Genomes of Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, Two Phylogenetically Distinct Probiotics.

    Science.gov (United States)

    Khatri, Indu; Sharma, Shailza; Ramya, T N C; Subramanian, Srikrishna

    2016-01-01

    Several spore-forming strains of Bacillus are marketed as probiotics due to their ability to survive harsh gastrointestinal conditions and confer health benefits to the host. We report the complete genomes of two commercially available probiotics, Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, and compare them with the genomes of other Bacillus and Lactobacillus. The taxonomic position of both organisms was established with a maximum-likelihood tree based on twenty six housekeeping proteins. Analysis of all probiotic strains of Bacillus and Lactobacillus reveal that the essential sporulation proteins are conserved in all Bacillus probiotic strains while they are absent in Lactobacillus spp. We identified various antibiotic resistance, stress-related, and adhesion-related domains in these organisms, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival during antibiotic treatment and harsh conditions.

  6. Isolated Bacterial Spores at High-velocity Survive Surface Impacts in Vacuum

    Science.gov (United States)

    Austin, Daniel; Barney, Brandon

    We present experiments in which bacterial spores were found to survive being accelerated in vacuum to velocities in the range 30-120 m/s and impacted on a dense target. In these experiments, spores of Bacillus subtilis spores were charged using electrospray at atmospheric pressure, dried, and then introduced into high vacuum. Through choice of skimmers and beam tubes, different velocity ranges were achieved. An image-charge detector observed the charged spores, providing total charge and velocity. The spores then impacted a glass target within a collection vessel. After the experiment, the collection vessel contents were extracted and cultured. Several positive and negative controls were used, including the use of antibiotic-resistant spores and antibiotic-containing (rifampicin) agar for culturing. These impact velocities are of particular interest for possible transport of bacterial spores from Mars to Phobos, and may have implications for planetary protection in a Phobos sample return mission. In addition, bacteria may reach similar velocities during a spacecraft crash (e.g., within components, or from spacecraft to surface materials during impact, etc.), raising concerns about forward contamination. The velocities of interest to transport of life between planets (panspermia) are somewhat higher, but these results complement shock-based experiments and contribute to the general discussion of impact survivability of organisms.

  7. Residual Agar Determination in Bacterial Spores by Electrospray Ionization Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Wahl, Karen L.; Colburn, Heather A.; Wunschel, David S.; Petersen, Catherine E.; Jarman, Kristin H.; Valentine, Nancy B.

    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 not 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.

  8. Residual agar determination in bacterial spores by electrospray ionization mass spectrometry.

    Science.gov (United States)

    Wahl, Karen L; Colburn, Heather A; Wunschel, David S; Petersen, Catherine E; Jarman, Kristin H; Valentine, Nancy B

    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-MS(n)) 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 nonirradiated, and not in the spores grown in broth. A sample containing approximately 10(8) 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 three false negatives for samples that were below the detection level of the method as documented.

  9. Deoxyribonucleic acid synthesis and deoxynucleotide metabolism during bacterial spore germination.

    Science.gov (United States)

    Setlow, P

    1973-06-01

    Deoxyribonucleic acid (DNA) synthesis during germination of Bacillus megaterium spores takes place in two stages. In stage I (0-55 min) DNA synthesis is slow and there is no detectable net synthesis, whereas in stage II (from 55 min on) the rate of synthesis is much faster and net DNA synthesis occurs. Deoxyribonucleotide pool sizes match the rates of DNA synthesis in stages I and II. The level of deoxyribonucleotide triphosphates is not correlated with the level of deoxyribonucleotide kinases, but rather with that of ribonucleotide reductase activity.

  10. Bacterial spores survive treatment with commercial sterilants and disinfectants.

    Science.gov (United States)

    Sagripanti, J L; Bonifacino, A

    1999-09-01

    This study compared the activity of commercial liquid sterilants and disinfectants on Bacillus subtilis spores deposited on three types of devices made of noncorrodible, corrodible, or polymeric material. Products like Renalin, Exspor, Wavicide-01, Cidexplus, and cupric ascorbate were tested under conditions specified for liquid sterilization. These products, at the shorter times indicated for disinfection, and popular disinfectants, like Clorox, Cavicide, and Lysol were also studied. Data obtained with a sensitive and quantitative test suggest that commercial liquid sterilants and disinfectants are less effective on contaminated surfaces than generally acknowledged.

  11. Indirect Detection Of Bacillus Anthracis (Anthrax) Using Amplified Gamma Phage-Based Assays

    Science.gov (United States)

    2007-11-01

    Figure 3.3 Pasteur Institute TEM of Bacillus surface 31 Bacillus anthracis is taxonomically aligned with B. cereus , B. thuringiensis and B...None of the DNA from bacteria (B. anthracis, B. cereus , Staphylococcus aureus, Pseudomonas aeroginosa, and Neisseria gonorrhea), yeast, blood , or...49-54. 59. Ryzhov, V., Y. Hathout, and C. Fenselau, Rapid Characterization of Spores of Bacillus Cereus Group Bacteria by Matrix-assisted Laser

  12. Investigation of bacterial spore structure by high resolution solid-state nuclear magnetic resonance spectroscopy and transmission electron microscopy.

    Science.gov (United States)

    Leuschner, R G; Lillford, P J

    2001-01-22

    High resolution solid-state nuclear magnetic resonance spectroscopy (NMR) in combination with transmission electron microscopy (TEM) of spores of Bacillus cereus, an outer coatless mutant B. subtilis 322, an inner coatless mutant B. subtilis 325 and of germinated spores of B. subtilis CMCC 604 were carried out. Structural differences in the coats, mainly protein of spores were reflected by NMR spectra which indicated also differences in molecular mobility of carbohydrates which was partially attributed to the cortex. Dipicolinic acid (DPA) of spores of B. cereus displayed a high degree of solid state order and may be crystalline. Heat activation was studied on spores of B. subtilis 357 lux + and revealed a structural change when analysed by TEM but this was not associated with increases in molecular mobility since no effects were measured by NMR.

  13. Effects of Endogenous d-Alanine Synthesis and Autoinhibition of Bacillus anthracis Germination on In Vitro and In Vivo Infections▿

    OpenAIRE

    McKevitt, Matthew T.; Bryant, Katie M.; Shakir, Salika M.; Larabee, Jason L.; Blanke, Steven R.; Lovchik, Julie; Lyons, C. Rick; Ballard, Jimmy D.

    2007-01-01

    Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous d-alanine production to the efficiency and timing of B. anthracis spore germination under in vit...

  14. Disinfection of Vegetative Cells of Bacillus anthracis

    Science.gov (United States)

    2016-03-01

    and the fate of vegetative cells resulting from augmented germination . In this study, data were generated on the inactivation of vegetative B...all the dilutions. First, a solution of 1000 mg chlorine solution was prepared in two steps . Sodium hypochlorite solution was diluted 1:5, and then 1... Germinant -Enhanced Decontamination of Bacillus Spores Adhered to Iron and Cement-Mortar Drinking Water Infrastructures. Appl. Environ. Microbiol. 2012, 78

  15. Spore Coat Architecture of Clostridium novyi-NT spores

    Energy Technology Data Exchange (ETDEWEB)

    Plomp, M; McCafferey, J; Cheong, I; Huang, X; Bettegowda, C; Kinzler, K; Zhou, S; Vogelstein, B; Malkin, A

    2007-05-07

    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. Towards this end, we have used both transmission electron microscopy and atomic force microscopy to determine the structure of dormant as well as 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.

  16. Analysis of germination capacity and germinant receptor (sub)clusters of genomesequenced Bacillus cereus environmental isolates and model strains

    NARCIS (Netherlands)

    Warda, Alicja K.; Xiao, Yinghua; Boekhorst, Jos; Wells-Bennik, Marjon H.J.; Nierop Groot, Masja N.; Abee, Tjakko

    2017-01-01

    Spore germination of 17 Bacillus cereus food isolates and reference strains was evaluated using flow cytometry analysis in combination with fluorescent staining at a single-spore level. This approach allowed for rapid collection of germination data under more than 20 conditions, including heat ac

  17. Heat stress leads to superoxide formation in Bacillus cereus detected using the fluorescent probe MitoSOX

    NARCIS (Netherlands)

    Mols, J.M.; Ceragioli, M.; Abee, T.

    2011-01-01

    Bacillus cereus is a food-borne human pathogen and food spoilage organism. Spores and vegetative cells of B. cereus can be found almost everywhere and therefore often end up in food processing equipment and food products. To remove spores and vegetative cells from food or equipment, harsh treatments

  18. Development of method for evaluating cell hardness and correlation between bacterial spore hardness and durability

    Directory of Open Access Journals (Sweden)

    Nakanishi Koichi

    2012-06-01

    Full Text Available Abstract Background Despite the availability of conventional devices for making single-cell manipulations, determining the hardness of a single cell remains difficult. Here, we consider the cell to be a linear elastic body and apply Young’s modulus (modulus of elasticity, which is defined as the ratio of the repulsive force (stress in response to the applied strain. In this new method, a scanning probe microscope (SPM is operated with a cantilever in the “contact-and-push” mode, and the cantilever is applied to the cell surface over a set distance (applied strain. Results We determined the hardness of the following bacterial cells: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and five Bacillus spp. In log phase, these strains had a similar Young’s modulus, but Bacillus spp. spores were significantly harder than the corresponding vegetative cells. There was a positive, linear correlation between the hardness of bacterial spores and heat or ultraviolet (UV resistance. Conclusions Using this technique, the hardness of a single vegetative bacterial cell or spore could be determined based on Young’s modulus. As an application of this technique, we demonstrated that the hardness of individual bacterial spores was directly proportional to heat and UV resistance, which are the conventional measures of physical durability. This technique allows the rapid and direct determination of spore durability and provides a valuable and innovative method for the evaluation of physical properties in the field of microbiology.

  19. Genomics, evolution, and crystal structure of a new family of bacterial spore kinases.

    Science.gov (United States)

    Scheeff, Eric D; Axelrod, Herbert L; Miller, Mitchell D; Chiu, Hsiu-Ju; Deacon, Ashley M; Wilson, Ian A; Manning, Gerard

    2010-05-01

    Bacterial spore formation is a complex process of fundamental relevance to biology and human disease. The spore coat structure is complex and poorly understood, and the roles of many of the protein components remain unclear. We describe a new family of spore coat proteins, the bacterial spore kinases (BSKs), and the first crystal structure of a BSK, YtaA (CotI) from Bacillus subtilis. BSKs are widely distributed in spore-forming Bacillus and Clostridium species, and have a dynamic evolutionary history. Sequence and structure analyses indicate that the BSKs are CAKs, a prevalent group of small molecule kinases in bacteria that is distantly related to the eukaryotic protein kinases. YtaA has substantial structural similarity to CAKs, but also displays distinctive features that broaden our understanding of the CAK group. Evolutionary constraint analysis of the protein surfaces indicates that members of the BSK family have distinct clade-conserved patterns in the substrate binding region, and probably bind and phosphorylate distinct targets. Several classes of BSKs have apparently independently lost catalytic activity to become pseudokinases, indicating that the family also has a major noncatalytic function.

  20. Bilirubin Oxidase Activity of Bacillus subtilis CotA

    OpenAIRE

    Sakasegawa, S; Ishikawa, H.; Imamura, S.; Sakuraba, H.; Goda, S.; Ohshima, T.

    2006-01-01

    The spore coat protein CotA from Bacillus subtilis was previously identified as a laccase. We have now found that CotA also shows strong bilirubin oxidase activity and markedly higher affinity for bilirubin than conventional bilirubin oxidase. This is the first characterization of bilirubin oxidase activity in a bacterial protein.

  1. Application of X-ray microscopy in food science investigation of high pressure affected bacterial spores

    Science.gov (United States)

    Mönch, Susanne; Heinz, Volker; Guttmann, Peter; Knorr, Dietrich

    2000-05-01

    Using the Göttingen transmission X-ray microscope at BESSY the effect of different pressure and temperature levels during the high hydrostatic pressure (HP) treatment was investigated. At 150 MPa and temperatures up to 50 °C the triggering of germination was observed by standard microbiological methods with Bacillus subtilis spores. Increasing the temperature to 70 °C at the same pressure level killed the spores without any indication of germination. By X-ray microscopy images it could be shown that the typical disintegration of the protoplast is inhibited. This suggests that the enzymic reaction pathway is possibly affected under specific pressure temperature conditions.

  2. Optimization of Spore Forming Bacteria Flooding for Enhanced Oil Recovery in North Sea Chalk Reservoir

    DEFF Research Database (Denmark)

    Halim, Amalia Yunita; Nielsen, Sidsel Marie; Eliasson Lantz, Anna;

    2015-01-01

    was used for this purpose. A spore forming bacterium, Bacillus licheniformis 421, was used as it was shown to be a good candidate in the previous study. Bacterial spore can penetrate deeper into the chalk rock, squeezing through the pore throats. Our results show that B. licheniformis 421 when injected...... as a secondary technique can recover 4% more of the original oil in place (OOIP) as compared with the seawater flooding. Furthermore, when applied as tertiary technique it can recover 1.4% OOIP of the residual oil. The effective permeability decreased in the first two sections of the core (0-1.2 cm and 1...

  3. Effects of meteorological conditions on spore plumes

    Science.gov (United States)

    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.

  4. Thermal Inactivation of Bacillus anthracis Spores Using Rapid Resistive Heating

    Science.gov (United States)

    2016-03-24

    setup including a laptop with appropriate software, a DC power supply, alligator clips attached to the power supply, a FLIR (forward looking infrared...filament, were tested on the microscope to validate the purity of tungsten. The samples included 4 light bulb filaments from different brands and the...easily clamped by the alligator clips, which are attached to the direct current power supply. The lamp cord was cut using wire snips in order to

  5. Identification of Bacillus strains for biological control of catfish pathogens.

    Science.gov (United States)

    Ran, Chao; Carrias, Abel; Williams, Malachi A; Capps, Nancy; Dan, Bui C T; Newton, Joseph C; Kloepper, Joseph W; Ooi, Ei L; Browdy, Craig L; Terhune, Jeffery S; Liles, Mark R

    2012-01-01

    Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×10(7) CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (Pbiological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture.

  6. Human Neutrophils Kill Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

  7. Human neutrophils kill Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Anne Mayer-Scholl

    2005-11-01

    Full Text Available Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

  8. Human neutrophils kill Bacillus anthracis.

    Science.gov (United States)

    Mayer-Scholl, Anne; Hurwitz, Robert; Brinkmann, Volker; Schmid, Monika; Jungblut, Peter; Weinrauch, Yvette; Zychlinsky, Arturo

    2005-11-01

    Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

  9. Monitoring biochemical changes in bacterial spore during thermal and pressure-assisted thermal processing using FT-IR spectroscopy.

    Science.gov (United States)

    Subramanian, Anand; Ahn, Juhee; Balasubramaniam, V M; Rodriguez-Saona, Luis

    2007-10-31

    Pressure-assisted thermal processing (PATP) is being widely investigated for processing low acid foods. However, its microbial safety has not been well established and the mechanism of inactivation of pathogens and spores is not well understood. Fourier transform infrared (FT-IR) spectroscopy was used to study some of the biochemical changes in bacterial spores occurring during PATP and thermal processing (TP). Spore suspensions (approximately 10(9) CFU/mL of water) of Clostridium tyrobutyricum, Bacillus sphaericus, and three strains of Bacillus amyloliquefaciens were treated by PATP (121 degrees C and 700 MPa) for 0, 10, 20, and 30 s and TP (121 degrees C) for 0, 10, 20, and 30 s. Treated and untreated spore suspensions were analyzed using FT-IR in the mid-infrared region (4000-800 cm(-1)). Multivariate classification models based on soft independent modeling of class analogy (SIMCA) were developed using second derivative-transformed spectra. The spores could be differentiated up to the strain level due to differences in their biochemical composition, especially dipicolinic acid (DPA) and secondary structure of proteins. During PATP changes in alpha-helix and beta-sheets of secondary protein were evident in the spectral regions 1655 and 1626 cm(-1), respectively. Infrared absorption bands from DPA (1281, 1378, 1440, and 1568 cm(-1)) decreased significantly during the initial stages of PATP, indicating release of DPA. During TP changes were evident in the bands associated with secondary proteins. DPA bands showed little or no change during TP. A correlation was found between the spore's Ca-DPA content and its resistance to PATP. FT-IR spectroscopy could classify different strains of bacterial spores and determine some of the changes occurring during spore inactivation by PATP and TP. Furthermore, this technique shows great promise for rapid screening PATP-resistant bacterial spores.

  10. Instruction for evaluating deposit of bacillus thuringiensis formulas during aerial treatments. Information report No. LAU-X-54

    Energy Technology Data Exchange (ETDEWEB)

    Smirnoff, W.A.

    1982-01-01

    Studies carried out form many years revealed that the methods used for deposit assessment of chemical insecticides could not be used with Bacillus thuringiensis. A new method was developed giving the quantity of viable spores dispersed per surface unit. Details of this method are concisely described in this document. It specifically provides instructions for evaluating deposit of Bacillus thuringiensis formulas during aerial treatments.

  11. Draft Genome Sequence of Bacillus pseudalcaliphilus PN-137T (DSM 8725), an Alkaliphilic Halotolerant Bacterium Isolated from Garden Soils.

    Science.gov (United States)

    Wang, Jie-Ping; Liu, Bo; Liu, Guo-Hong; Xiao, Rong-Feng; Zheng, Xue-Fang; Shi, Huai; Ge, Ci-Bin

    2015-01-01

    Bacillus pseudalcaliphilus PN-137(T) (DSM 8725) is a Gram-positive, spore-forming, alkaliphilic, and halotolerant bacterium. Here, we report the 4.49-Mb genome sequence of B. pseudalcaliphilus PN-137(T), which will accelerate the application of this alkaliphile and provide useful information for genomic taxonomy and phylogenomics of Bacillus-like bacteria.

  12. Long-term survival of bacterial spores in space

    Science.gov (United States)

    Horneck, G.; Bucker, H.; Reitz, G.

    1994-01-01

    On board of the NASA Long Duration Exposure Facility (LDEF), spores of Bacillus subtilis in monolayers (10(exp 6)/sample) or multilayers (10(exp 8)/sample) were exposed to the space environment for nearly six years and their survival was analyzed after retrieval. The response to space parameters, such as vacuum (10(exp -6) Pa), solar electromagnetic radiation up to the highly energetic vacuum-ultraviolet range 10(exp 9) J/sq m) and/or cosmic radiation (4.8 Gy), was studied and compared to the results of a simultaneously running ground control experiment. If shielded against solar ultraviolet (UV)-radiation, up to 80% of spores in multilayers survive in space. Solar UV-radiation, being the most deleterious parameter of space, reduces survival by 4 orders of magnitude or more. However, up to 10(exp 4) viable spores were still recovered, even in completely unprotected samples. Substances, such as glucose or buffer salts serve as chemical protectants. With this 6 year study in space, experimental data are provided to the discussion on the likelihood of 'Panspermia'.

  13. Measuring Total and Germinable Spore Populations

    Science.gov (United States)

    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.

  14. Impact of two DNA repair pathways, homologous recombination and non-homologous end joining, on bacterial spore inactivation under simulated martian environmental conditions

    Science.gov (United States)

    Moeller, Ralf; Schuerger, Andrew C.; Reitz, Günther; Nicholson, Wayne L.

    2011-09-01

    Spores of Bacillus subtilis were used as a model system to study the impact of the two major DNA double-strand break (DSB) repair mechanisms [homologous recombination (HR) and non-homologous end-joining (NHEJ)] on the survivability of air-dried mono- and multilayers of bacterial spores under a simulated martian environment; i.e., an environment with low temperature (-10 °C), pure CO 2 atmosphere (99.99% CO 2), 200-1100 nm UV-VIS-NIR radiation, and 0.69 kPa pressure. Spores in multilayers exhibited low inactivation rates compared to monolayers, mainly due to shadowing effects of overlying spores. Simulated martian UV irradiation reduced dramatically spore viability, whereas when shielded from martian UV radiation, spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to simulated martian environmental conditions than were wild-type spores. In addition, NHEJ-deficient spores were consistently more sensitive than HR-deficient spores to simulated Mars environmental conditions, suggesting that DSBs were an important type of DNA damage. The results indicated that both HR and NHEJ provide an efficient set of DNA repair pathways ensuring spore survival after exposure to simulated martian environmental conditions.

  15. Analysis of Alicyclobacillus acidoterrestris spores from different sporulation media subjected to wet-heat

    Directory of Open Access Journals (Sweden)

    Celenk Molva

    2015-12-01

    Full Text Available The presence of Alicyclobacillus acidoterrestris endospores in fruit juices is a significant problem for the juice industry since they are able to survive pasteurization subsequently leading to the spoilage. To evaluate the mechanism of wet-heat, structural damage and the leakages of intracellular materials of A. acidoterrestris DSM 3922 spores from different sporulation media was studied at 90°C (15-45 min. For sporulation, Bacillus acidoterrestris agar, Bacillus acidocaldarius agar, potato dextrose agar and malt extract agar were used. Based on the scanning electron microscopy, loss of internal volume and structural integrity were observed following heating which were further confirmed by the leakages of intracellular components. The obtained results suggest that the inactivation of A. acidoterrestris DSM 3922 spores by wet-heat is associated with damage to the coat and inner membrane depending on the sporulation medium composition and heating time.

  16. Fluorescence-based methods for the detection of pressure-induced spore germination and inactivation

    Science.gov (United States)

    Baier, Daniel; Reineke, Kai; Doehner, Isabel; Mathys, Alexander; Knorr, Dietrich

    2011-03-01

    The application of high pressure (HP) provides an opportunity for the non-thermal preservation of high-quality foods, whereas highly resistant bacterial endospores play an important role. It is known that the germination of spores can be initiated by the application of HP. Moreover, the resistance properties of spores are highly dependent on their physiological states, which are passed through during the germination. To distinguish between different physiological states and to detect the amount of germinated spores after HP treatments, two fluorescence-based methods were applied. A flow cytometric method using a double staining with SYTO 16 as an indicator for germination and propidium iodide as an indicator for membrane damage was used to detect different physiological states of the spores. During the first step of germination, the spore-specific dipicolinic acid (DPA) is released [P. Setlow, Spore germination, Curr. Opin. Microbiol. 6 (2003), pp. 550-556]. DPA reacts with added terbium to form a distinctive fluorescent complex. After measuring the fluorescence intensity at 270 nm excitation wavelength in a fluorescence spectrophotometer, the amount of germinated spores can be determined. Spores of Bacillus subtilis were treated at pressures from 150 to 600 MPa and temperatures from 37 °C to 60 °C in 0.05 M ACES buffer solution (pH 7) for dwell times of up to 2 h. During the HP treatments, inactivation up to 2log 10 cycles and thermal sensitive populations up to 4log 10 cycles could be detected by plate counts. With an increasing number of thermal sensitive spores, an increased proportion of spores in germinated states was detected by flow cytometry. Also the released amount of DPA increased during the dwell times. Moreover, a clear pressure-temperature-time-dependency was shown by screening different conditions. The fluorescence-based measurement of the released DPA can provide the opportunity of an online monitoring of the germination of spores under HP inside

  17. Spore test parameters matter: Mesophilic and thermophilic spore counts detected in raw milk and dairy powders differ significantly by test method.

    Science.gov (United States)

    Kent, D J; Chauhan, K; Boor, K J; Wiedmann, M; Martin, N H

    2016-07-01

    United States dairy industry exports have steadily risen in importance over the last 10yr, with dairy powders playing a particularly critical role. Currently, approximately half of US-produced nonfat dry milk and skim milk powder is exported. Reaching new and expanding existing export markets relies in part on the control of endospore-forming bacteria in dairy powders. This study reports baseline mesophilic and thermophilic spore counts and spore populations from 55 raw material samples (primarily raw milk) and 33 dairy powder samples from dairy powder processors across the United States. Samples were evaluated using various spore testing methodologies and included initial heat treatments of (1) 80°C for 12 min; (2) 100°C for 30 min; and (3) 106°C for 30 min. Results indicate that significant differences in both the level and population of spores were found for both raw milk and dairy powders with the various testing methods. Additionally, on average, spore counts were not found to increase significantly from the beginning to the end of dairy powder processing, most likely related to the absence of biofilm formation by processing plant-associated sporeformers (e.g., Anoxybacillus sp.) in the facilities sampled. Finally, in agreement with other studies, Bacillus licheniformis was found to be the most prevalent sporeformer in both raw materials and dairy powders, highlighting the importance of this organism in developing strategies for control and reduction of spore counts in dairy powders. Overall, this study emphasizes the need for standardization of spore enumeration methodologies in the dairy powder industry.

  18. Microarray Analysis of Transposon Insertion Mutants in Bacillus Anthracis: Global Identification of Genes Required for Sporulation and Germination

    Science.gov (United States)

    2007-02-01

    Gilois, M. Rose, and D. Lereclus. 2001. Oligopep- tide permease is required for expression of the Bacillus thuringiensis plcR regulon and for...non- toxin gene expression in Bacillus anthracis. Infect. Immun. 65:3091–3099. 10. Ikeda, R. A., C. M. Ligman, and S. Warshamana. 1992. T7 promoter con...nontoxi- genic Bacillus anthracis spore vaccines based on strains expressing mutant vari- ants of lethal toxin components. Vaccine 23:5688–5697. 17. Read

  19. Enhanced specificity of bacterial spore identification by oxidation and mass spectrometry.

    Science.gov (United States)

    Demirev, Plamen A

    2004-01-01

    Addition of an oxidizing agent (e.g., hydrogen peroxide) to intact spores selectively and completely oxidizes Met-containing biomarker proteins by formation of Met sulfoxides. This reaction increases the masses of the biomarker proteins observed in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of Bacillus spores by Deltam = (16 x n) Da, where n is the number of Met residues in the sequence of each individual protein. The procedure is very rapid, and can be performed in situ (i.e., on the MALDI target). It confirms the identity of individual biomarkers by comparing the number of Met amino acids from the experimentally determined mass shifts with predictions for n from the tentative amino acid sequence for each protein. In turn, accurate determination of n for several biomarkers allows rapid validation of the initial spore identification by MALDI-MS.

  20. Spectral refractive index and extinction cross-section of BG spores

    Science.gov (United States)

    Airola, Marc B.; Boggs, Nathan T.; Jackman, Joany; Fainchtein, Raul; Carter, Christopher C.; Thomas, Michael E.

    2005-05-01

    Despite the wide spread need for optical cross-section data on single spore bio-aerosols, available databases are sparse and unreliable. Information reported is based on short path measurements on high concentration media containing particle clusters. This represents an upper bound to the single spore cross-section. Measurements on single spore aerosolized media demand long path lengths and moderate particle concentration. Transmittance measurements need to be in the single scatter limit as well. These requirements are often difficult to meet. We present a procedure that leads to aerosol extinction and backscatter cross-sections in a straightforward manner. Transmittance measurements of thin films of bio-aerosols are used to obtain the bulk refractive index. This result and the measured size distribution can be used in a T-matrix calculation to yield the desired cross-sections. To illustrate this technique, infrared cross-sections are obtained for Bacillus globigii.

  1. Molecular Kinetics of Reviving Bacterial Spores

    OpenAIRE

    2013-01-01

    Bacterial spores can remain dormant for years, yet they possess a remarkable potential to rapidly resume a vegetative life form. Here, we identified a distinct phase at the onset of spore outgrowth, designated the ripening period. This transition phase is exploited by the germinating spore for molecular reorganization toward elongation and subsequent cell division. We have previously shown that spores of different ages, kept under various temperatures, harbor dissimilar molecular reservoirs (...

  2. A selective chromogenic agar that distinguishes Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis.

    Science.gov (United States)

    Juergensmeyer, Margaret A; Gingras, Bruce A; Restaino, Lawrence; Frampton, Elon W

    2006-08-01

    A selective and differential plating medium, R & F anthracis chromogenic agar (ACA), has been developed for isolating and identifying presumptive colonies of Bacillus anthracis. ACA contains the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-choline phosphate that upon hydrolysis yields teal (blue green) colonies indicating the presence of phosphatidylcholine-specific phospholipase C (PC-PLC) activity. Among seven Bacillus species tested on ACA, only members of the Bacillus cereus group (B. anthracis, B. cereus, and B. thuringiensis) produced teal colonies (PC-PLC positive) having cream rings. Examination of colony morphology in 18 pure culture strains of B. anthracis (15 ATCC strains plus AMES-1-RIID, ANR-1, and AMED-RIID), with one exception, required 48 h at 35 to 37 degrees C for significant color production, whereas only 24 h was required for B. cereus and B. thuringiensis. This differential rate of PC-PLC synthesis in B. anthracis (due to the truncated plcR gene and PlcR regulator in B. anthracis) allowed for the rapid differentiation on ACA of presumptive colonies of B. anthracis from B. cereus and B. thuringiensis in both pure and mixed cultures. Effective recovery of B. anthracis from a variety of matrices having both high (soil and sewage) and low microbial backgrounds (cloth, paper, and blood) spiked with B. anthracis ANR-1 spores suggests the probable utility of ACA plating for B. anthracis recovery in a diversity of applications.

  3. UV-Resistant Non-Spore-Forming Bacteria From Spacecraft-Assembly Facilities

    Science.gov (United States)

    Venkateswaran, Kasthuri

    2008-01-01

    Four species of non-spore-forming bacteria collected from clean-room surfaces in spacecraft-assembly facilities could survive doses of ultraviolet (UV) radiation that would suffice to kill most known cultivable bacterial species. In a previous study, high UV resistance was found in spores of the SAFR-032 strain of Bacillus pumilus, as reported in "Ultraviolet- Resistant Bacterial Spores," NASA Tech Briefs, Vol. 31, No. 9 (September 2007), page 94. These studies are parts of a continuing effort to understand the survival of hardy species of bacteria under harsh conditions, and develop means of sterilizing spacecraft to prevent biocontamination of Mars that could in turn interfere with future life detection missions. The four species investigated were Arthrobacter sp. KSC_Ak2i, Microbacterium schleiferi LMA_AkK1, Brevundimonas diminuta KSC_Ak3a, and Sphingomonas trueperi JSC_Ak7-3. In the study, cells of these species were mixed into Atacama Desert soil (to elucidate the shadowing effect of soil particles) and the resulting mixtures were tested both in solution and in a desiccated state under simulated Martian atmospheric and UV conditions. The UV-survival indices of Arthrobacter sp. and Microbacterium schleiferi were found to be comparable to those of Bacillus pumilus spores.

  4. Germination and conjugation of Bacillus thuringiensis subsp. israelensis in the intestine of gnotobiotic rats

    DEFF Research Database (Denmark)

    Wilcks, Andrea; Ørum-Smidt, Lasse; Bahl, Martin Iain;

    2008-01-01

    Aims: To study the ability of Bacillus thuringiensis subsp. israelensis spores to germinate and subsequently transfer a conjugative plasmid in the intestinal tract of gnotobiotic rats. Methods and Results: Germination was studied by feeding germ-free rats with spores of a B. thuringiensis strain...... the conjugative plasmid pXO16 was introduced. Both strains were given as spores and transfer of pXO16 was observed from the donor to the recipient strain. Conclusions: Bacillus thuringiensis is able to have a full life cycle in the intestine of gnotobiotic rats including germination of spores, several cycles...... harbouring a plasmid encoding green fluorescent protein (GFP), which enabled quantification of germinated bacteria by flow cytometry. To study in vivo conjugation, germ-free rats were first associated with a B. thuringiensis recipient strain and after 1 week an isogenic donor strain harbouring...

  5. Survival and conjugal transfer between Bacillus thuringiensis strains in aquatic environment

    OpenAIRE

    Furlaneto Luciana; Saridakis Halha Ostrensky; Arantes Olívia Márcia Nagy

    2000-01-01

    Field and laboratory studies were conducted to assess the survival of cells and spores and plasmid transfer between Bacillus thuringienis strains in aquatic environment. Results indicated that cells and spores of B. thuringiensis can survive for 10 days in water, without altering their number. The sporulation process began after 12-15 hours of inoculation of water. B. thuringiensis was able to transfer conjugative plasmids in the aquatic environment.

  6. Comparative genome analysis of Bacillus cereus group genomes withBacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Iain; Sorokin, Alexei; Kapatral, Vinayak; Reznik, Gary; Bhattacharya, Anamitra; Mikhailova, Natalia; Burd, Henry; Joukov, Victor; Kaznadzey, Denis; Walunas, Theresa; D' Souza, Mark; Larsen, Niels; Pusch,Gordon; Liolios, Konstantinos; Grechkin, Yuri; Lapidus, Alla; Goltsman,Eugene; Chu, Lien; Fonstein, Michael; Ehrlich, S. Dusko; Overbeek, Ross; Kyrpides, Nikos; Ivanova, Natalia

    2005-09-14

    Genome features of the Bacillus cereus group genomes (representative strains of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis sub spp israelensis) were analyzed and compared with the Bacillus subtilis genome. A core set of 1,381 protein families among the four Bacillus genomes, with an additional set of 933 families common to the B. cereus group, was identified. Differences in signal transduction pathways, membrane transporters, cell surface structures, cell wall, and S-layer proteins suggesting differences in their phenotype were identified. The B. cereus group has signal transduction systems including a tyrosine kinase related to two-component system histidine kinases from B. subtilis. A model for regulation of the stress responsive sigma factor sigmaB in the B. cereus group different from the well studied regulation in B. subtilis has been proposed. Despite a high degree of chromosomal synteny among these genomes, significant differences in cell wall and spore coat proteins that contribute to the survival and adaptation in specific hosts has been identified.

  7. Isolating and Purifying Clostridium difficile Spores

    Science.gov (United States)

    Edwards, Adrianne N.; McBride, Shonna M.

    2016-01-01

    Summary The ability for the obligate anaerobe, Clostridium difficile, to form a metabolically dormant spore is critical for the survival of this organism outside of the host. This spore form is resistant to a myriad of environmental stresses, including heat, desiccation and exposure to disinfectants and antimicrobials. These intrinsic properties of spores allow C. difficile to survive long-term in an oxygenated environment, to be easily transmitted from host-to-host and to persist within the host following antibiotic treatment. Because of the importance of the spore form to the C. difficile lifecycle and treatment and prevention of C. difficile infection (CDI), the isolation and purification of spores are necessary to study the mechanisms of sporulation and germination, investigate spore properties and resistances, and for use in animal models of CDI. This chapter provides basic protocols, in vitro growth conditions and additional considerations for purifying C. difficile spores for a variety of downstream applications. PMID:27507337

  8. Ultra-violet-resistant mutants of Bacillus thuringiensis

    Energy Technology Data Exchange (ETDEWEB)

    Jones, D.R.; Karunakaran, V. (Polytechnic of Central London (UK). Faculty of Engineering and Science, School of Biological and Health Sciences); Burges, H.D. (Institute of Horticultural Research, Littlehampton (UK)); Hacking, A.J. (Reading Univ. (UK). Dextra Labs.Ltd.)

    1991-06-01

    One of the main disadvantages of using Bacillus thuringiensis as an insecticide is that the spore and crystal preparations applied to foliage are readily washed away by rain and are inactivated by sunlight. Spores from some strains of B. thuringiensis have been shown to be highly sensitive to u.v. light. This study has demonstrated how mutants with increased resistance to u.v., isolated by successive rounds of u.v. irradiation, and additionally with increased specific pathogenicity can be isolated. These techniques should be applied to strains that are frequently used in the industrial production of B.thuringiensis toxin. (author).

  9. Role of fatty acids in Bacillus environmental adaptation

    Directory of Open Access Journals (Sweden)

    Sara Esther Diomande

    2015-08-01

    Full Text Available The large bacterial genus genus Bacillus is widely distributed in the environment and is able to colonize highly diverse niches. Some Bacillus species harbour pathogenic characteristics. The fatty acid (FA composition is among the essential criteria used to define Bacillus species. Some elements of the FA pattern composition are common to Bacillus species, whereas others are specific and can be categorized in relation to the ecological niches of the species. Bacillus species are able to modify their FA patterns to adapt to a wide range of environmental changes, including changes in the growth medium, temperature, food processing conditions, and pH. Like many other Gram-positive bacteria, Bacillus strains display a well-defined FA synthesis II system that is equilibrated with a FA degradation pathway and regulated to efficiently respond to the needs of the cell. Like endogenous FAs, exogenous FAs may positively or negatively affect the survival of Bacillus vegetative cells and the spore germination ability in a given environment. Some of these exogenous FAs may provide a powerful strategy for preserving food against contamination by the Bacillus pathogenic strains responsible for foodborne illness.

  10. Sensing and inactivation of Bacillus anthracis Sterne by polymer-bromine complexes.

    Science.gov (United States)

    D'Angelo, Paola A; Bromberg, Lev; Hatton, T Alan; Wilusz, Eugene

    2016-08-01

    We report on the performance of brominated poly(N-vinylpyrrolidone) (PVP-Br), brominated poly(ethylene glycol) (PEG-Br), and brominated poly(allylamine-co-4-aminopyridine) (PAAm-APy-Br) for their ability to decontaminate Bacillus anthracis Sterne spores in solution while also allowing for the sensing of the spores. The polymers were brominated by bromine using carbon tetrachloride or potassium tribromide as solvents, with bromine loadings ranging from 1.6 to 4.2 mEq/g of polymer. B. anthracis Sterne spores were exposed to increasing concentrations of brominated polymers for 5 min, while the kinetics of the sporicidal activity was assessed. All brominated polymers demonstrated spore log-kills of 8 within 5 min of exposure at 12 mg/mL aqueous polymer concentration. Sensing of spores was accomplished by measuring the release of dipicolinic acid (DPA) from the spore using time-resolved fluorescence. Parent, non-brominated polymers did not cause any release of DPA and the spores remained viable. In contrast, spores exposed to the brominated polymers were inactivated and the release of DPA was observed within minutes of exposure. Also, this release of DPA continued for a long time after spore inactivation as in a controlled release process. The DPA release was more pronounced for spores exposed to brominated PVP and brominated PEG-8000 compared to brominated PAAm-APy and brominated PEG-400. Using time-resolved fluorescence, we detected as low as 2500 B. anthracis spores, with PEG-8000 being more sensitive to low spore numbers. Our results suggest that the brominated polymers may be used effectively as decontamination agents against bacterial spores while also providing the sensing capability.

  11. Toxigenic potential and heat survival of spore-forming bacteria isolated from bread and ingredients.

    Science.gov (United States)

    De Bellis, Palmira; Minervini, Fiorenza; Di Biase, Mariaelena; Valerio, Francesca; Lavermicocca, Paola; Sisto, Angelo

    2015-03-16

    Fifty-four spore-forming bacterial strains isolated from bread ingredients and bread, mainly belonging to the genus Bacillus (including Bacillus cereus), together with 11 reference strains were investigated to evaluate their cytotoxic potential and heat survival in order to ascertain if they could represent a risk for consumer health. Therefore, we performed a screening test of cytotoxic activity on HT-29 cells using bacterial culture filtrates after growing bacterial cells in Brain Heart Infusion medium and in the bread-based medium Bread Extract Broth (BEB). Moreover, immunoassays and PCR analyses, specifically targeting already known toxins and related genes of B. cereus, as well as a heat spore inactivation assay were carried out. Despite of strain variability, the results clearly demonstrated a high cytotoxic activity of B. cereus strains, even if for most of them it was significantly lower in BEB medium. Cytotoxic activity was also detected in 30% of strains belonging to species different from B. cereus, although, with a few exceptions (e.g. Bacillus simplex N58.2), it was low or very low. PCR analyses detected the presence of genes involved in the production of NHE, HBL or CytK toxins in B. cereus strains, while genes responsible for cereulide production were not detected. Production of NHE and HBL toxins was also confirmed by specific immunoassays only for B. cereus strains even if PCR analyses revealed the presence of related toxin genes also in some strains of other species. Viable spore count was ascertained after a heat treatment simulating the bread cooking process. Results indicated that B. amyloliquefaciens strains almost completely survived the heat treatment showing less than 2 log-cycle reductions similarly to two strains of B. cereus group III and single strains belonging to Bacillus subtilis, Bacillus mojavensis and Paenibacillus spp. Importantly, spores from strains of the B. cereus group IV exhibited a thermal resistance markedly lower than B

  12. Analysis of the Raman spectra of Ca(2+)-dipicolinic acid alone and in the bacterial spore core in both aqueous and dehydrated environments.

    Science.gov (United States)

    Kong, Lingbo; Setlow, Peter; Li, Yong-qing

    2012-08-21

    The core of dormant bacterial spores suspended in water contains a large depot of dipicolinic acid (DPA) chelated with divalent cations, predominantly Ca(2+) (CaDPA), and surrounded by water molecules. Since the intensities of the vibration bands of CaDPA molecules depend significantly on the water content in the CaDPA's environment, the Raman spectra of CaDPA in spores may allow the determination of the spore core's hydration state. We have measured Raman spectra of single spores of three Bacillus species in different hydration states including the spores suspended in water, air-dried and vacuum-dried. As a comparison, we also measured the Raman spectra of CaDPA and DPA in different forms including in aqueous solution, and as amorphous powder and crystalline form. We also monitored changes in Raman spectra of an individual spore during dehydration under vacuum. The results indicated that (1) the state of CaDPA in the core of a spore suspended in water is close to an amorphous solid or a glassy state, but still mixed with water molecules; (2) the ratio of intensities of Raman bands at 1575 and 1017 cm(-1) (I(1575)/I(1017)) is sensitive to the water content in the CaDPA's environment; (3) variations in I(1575)/I(1017) are small (∼4%) in a population of dormant Bacillus spores suspended in water; and (4) the I(1575)/I(1017) ratio increases significantly during dehydration under vacuum. Consequently, measurement of the I(1575)/I(1017) ratio of CaDPA in spores may allow a qualitative estimation of the degree of hydration of the bacterial spore's core.

  13. Bacterial spore heat resistance correlated with water content, wet density, and protoplast/sporoplast volume ratio.

    Science.gov (United States)

    Beaman, T C; Greenamyre, J T; Corner, T R; Pankratz, H S; Gerhardt, P

    1982-05-01

    Five types of dormant Bacillus spores, between and within species, were selected representing a 600-fold range in moist-heat resistance determined as a D100 value. The wet and dry density and the solids and water content of the entire spore and isolated integument of each type were determined directly from gram masses of material, with correction for interstitial water. The ratio between the volume occupied by the protoplast (the structures bounded by the inner pericytoplasm membrane) and the volume occupied by the sporoplast (the structures bounded by the outer pericortex membrane) was calculated from measurements made on electron micrographs of medially thin-sectioned spores. Among the various spore types, an exponential increase in the heat resistance correlated directly with the wet density and inversely with the water content and with the protoplast/sporoplast volume ratio. Altogether with results supported a hypothesis that the extent of heat resistance is based in whole or in part on the extent of dehydration and diminution of the protoplast in the dormant spore, without implications about physiological mechanisms for attaining this state.

  14. Decontamination Efficacy and Skin Toxicity of Two Decontaminants against Bacillus anthracis.

    Science.gov (United States)

    Stratilo, Chad W; Crichton, Melissa K F; Sawyer, Thomas W

    2015-01-01

    Decontamination of bacterial endospores such as Bacillus anthracis has traditionally required the use of harsh or caustic chemicals. The aim of this study was to evaluate the efficacy of a chlorine dioxide decontaminant in killing Bacillus anthracis spores in solution and on a human skin simulant (porcine cadaver skin), compared to that of commonly used sodium hypochlorite or soapy water decontamination procedures. In addition, the relative toxicities of these decontaminants were compared in human skin keratinocyte primary cultures. The chlorine dioxide decontaminant was similarly effective to sodium hypochlorite in reducing spore numbers of Bacillus anthracis Ames in liquid suspension after a 10 minute exposure. After five minutes, the chlorine dioxide product was significantly more efficacious. Decontamination of isolated swine skin contaminated with Bacillus anthracis Sterne with the chlorine dioxide product resulted in no viable spores sampled. The toxicity of the chlorine dioxide decontaminant was up to two orders of magnitude less than that of sodium hypochlorite in human skin keratinocyte cultures. In summary, the chlorine dioxide based decontaminant efficiently killed Bacillus anthracis spores in liquid suspension, as well as on isolated swine skin, and was less toxic than sodium hypochlorite in cultures of human skin keratinocytes.

  15. Bacillus anthracis Bioterrorism Incident, Kameido, Tokyo, 1993

    Science.gov (United States)

    Keim, Paul; Kaufmann, Arnold F.; Keys, Christine; Smith, Kimothy L.; Taniguchi, Kiyosu; Inouye, Sakae; Kurata, Takeshi

    2004-01-01

    In July 1993, a liquid suspension of Bacillus anthracis was aerosolized from the roof of an eight-story building in Kameido, Tokyo, Japan, by the religious group Aum Shinrikyo. During 1999 to 2001, microbiologic tests were conducted on a liquid environmental sample originally collected during the 1993 incident. Nonencapsulated isolates of B. anthracis were cultured from the liquid. Multiple-locus, variable-number tandem repeat analysis found all isolates to be identical to a strain used in Japan to vaccinate animals against anthrax, which was consistent with the Aum Shinrikyo members’ testimony about the strain source. In 1999, a retrospective case-detection survey was conducted to identify potential human anthrax cases associated with the incident, but none were found. The use of an attenuated B. anthracis strain, low spore concentrations, ineffective dispersal, a clogged spray device, and inactivation of the spores by sunlight are all likely contributing factors to the lack of human cases. PMID:15112666

  16. Bacterial spore detection and analysis using hyperpolarized (129)Xe chemi