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Sample records for bacillus atrophaeus spores

  1. 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.

  2. Cryogenic Irradiation of Bacillus Atrophaeus spores to understand microbial survival on Icy Bodies

    Science.gov (United States)

    Yerby, C. J.; Noell, A. C.; Hodyss, R. P.; Johnson, P. V.; Ponce, A.

    2017-12-01

    Bacterial Spores are useful indicator organisms for studying the survival of microbes and degradation of biomolecules on the surface of planetary icy bodies. To predict the limits of life's proliferation in space, specifically on icy bodies, it is essential to understand the ability of microbes to withstand photon and particle irradiation at cryogenic temperatures. Bacillus Atrophaeus spores were transferred onto stainless steel coupons by varied processes and subsequently frozen at Europan temperatures (16oK—273oK) in a vacuum at 8.7x10-8 Torr. An argon lamp bombarded the spore-containing coupons with a solar-like radiation spectra for a variety of times, and spores were removed from the coupons and enumerated in culture. To date, (n=43) coupons have been analyzed for spore kill-rates with regards to ice temperature and radiation exposure time. Results will be presented on the effect of cryogenic temperatures in improving radiation resistance of bacterial spores. This works also details methodology improvements by comparing different spore deposition and recovery methods before and after cryogenic irradiation.

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

    International Nuclear Information System (INIS)

    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.

    2013-01-01

    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

  4. 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.

  5. Influence of high voltage atmospheric cold plasma process parameters and role of relative humidity on inactivation of Bacillus atrophaeus spores inside a sealed package.

    Science.gov (United States)

    Patil, S; Moiseev, T; Misra, N N; Cullen, P J; Mosnier, J P; Keener, K M; Bourke, P

    2014-11-01

    Non-thermal plasma has received much attention for elimination of microbial contamination from a range of surfaces. This study aimed to determine the effect of a range of dielectric barrier discharge high voltage atmospheric cold plasma (HVACP) parameters for inactivation of Bacillus atrophaeus spores inside a sealed package. A sterile polystyrene Petri dish containing B. atrophaeus spore strip (spore population 2.3 × 10(6)/strip i.e. 6.36 log10/strip) was placed in a sealed polypropylene container and was subjected to HVACP treatment. The HVACP discharge was generated between two aluminium plate electrodes using a high voltage of 70kVRMS. The effects of process parameters, including treatment time, mode of exposure (direct/indirect), and working gas types, were evaluated. The influence of relative humidity on HVACP inactivation efficacy was also assessed. The inactivation efficacy was evaluated using colony counts. Optical absorption spectroscopy (OAS) was used to assess gas composition following HVACP exposure. A strong effect of process parameters on inactivation was observed. Direct plasma exposure for 60s resulted in ≥6 log10 cycle reduction of spores in all gas types tested. However, indirect exposure for 60s resulted in either 2.1 or 6.3 log10 cycle reduction of spores depending on gas types used for HVACP generation. The relative humidity (RH) was a critical factor in bacterial spore inactivation by HVACP, where a major role of plasma-generated species other than ozone was noted. Direct and indirect HVACP exposure for 60s at 70% RH recorded 6.3 and 5.7 log10 cycle reduction of spores, respectively. In summary, a strong influence of process parameters on spore inactivation was noted. Rapid in-package HVACP inactivation of bacterial spores within 30-60s demonstrates the promising potential application for reduction of spores on medical devices and heat-sensitive materials. Copyright © 2014 The Healthcare Infection Society. All rights reserved.

  6. 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.

  7. Inactivation of Bacillus atrophaeus by OH radicals

    Science.gov (United States)

    Ono, Ryo; Yonetamari, Kenta; Tokumitsu, Yusuke; Yonemori, Seiya; Yasuda, Hachiro; Mizuno, Akira

    2016-08-01

    The inactivation of Bacillus atrophaeus by OH radicals is measured. This study aims to evaluate the bactericidal effects of OH radicals produced by atmospheric-pressure nonthermal plasma widely used for plasma medicine; however, in this study, OH radicals are produced by vacuum ultraviolet (VUV) photolysis of water vapor instead of plasma to allow the production of OH radicals with almost no other reactive species. A 172 nm VUV light from a Xe2 excimer lamp irradiates a He-H2O mixture flowing in a quartz tube to photodissociate H2O to produce OH, H, O, HO2, H2O2, and O3. The produced reactive oxygen species (ROS) flow out of the quartz tube nozzle to the bacteria on an agar plate and cause inactivation. The inactivation by OH radicals among the six ROS is observed by properly setting the experimental conditions with the help of simulations calculating the ROS densities. A 30 s treatment with approximately 0.1 ppm OH radicals causes visible inactivation.

  8. The inactivation and removal of airborne Bacillus atrophaeus endospores from air circulation systems using UVC and HEPA filters.

    Science.gov (United States)

    Luna, V A; Cannons, A C; Amuso, P T; Cattani, J

    2008-02-01

    To (i) evaluate the UV radiation in the 'C' band/high efficient particulate air (UVC/HEPA) instrument's potential to inactivate spores of Bacillus atrophaeus and selected Bacillus species and (ii) test whether a titanium dioxide coating inside the cylindrical HEPA filter improves the system's efficacy. Known amounts of dried spore preparations of B. atrophaeus, Bacillus cereus, Bacillus megaterium, Bacillus stearothermophilus and Bacillus thuringiensis were exposed to the UVC lamp within a cylindrical HEPA filter for different time lengths (30 min to 48 h) and with different air flow speeds (0-235 l s(-1)). The log(10) reduction (range 5-16 logs) of colony forming units for spores exposed to the UVC compared with the unexposed spores was significant (P HEPA filter significantly increased the inactivation of spores (P HEPA unit could inactivate spores of B. atrophaeus, B. cereus, B. megaterium, B. stearothermophilus and B. thuringiensis. The UVC/HEPA unit represents an effective method of decontaminating circulating air within an air-duct system as found in a building.

  9. Phosphorescence In Bacillus Spores

    National Research Council Canada - National Science Library

    Reinisch, Lou; Swartz, Barry A; Bronk, Burt V

    2003-01-01

    .... Our present work attempts to build on this approach for environmental applications. We have measured a change in the fluorescence spectra of suspensions of Bacillus bacteria between the vegetative bacteria and their spores at room temperature...

  10. Genomic Signatures of Strain Selection and Enhancement in Bacillus atrophaeus var. globigii, a Historical Biowarfare Simulant

    Science.gov (United States)

    2011-03-25

    B_amyloliq – B. amyloliquefaciens ; B_NRRL - Bacillus . spp. NRRL; B_SG-1 – Bacillus spp. SG-1; B_thur – B. thuringiensis strains Al Hakam and var...Genomic Signatures of Strain Selection and Enhancement in Bacillus atrophaeus var. globigii, a Historical Biowarfare Simulant Henry S. Gibbons1...States of America, 11 Excet Inc., Aberdeen Proving Ground, Maryland, United States of America Abstract Background: Despite the decades-long use of Bacillus

  11. 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.

  12. 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.

  13. Inactivation of Bacillus atrophaeus and of Aspergillus niger using beams of argon ions, of oxygen molecules and of oxygen atoms

    Energy Technology Data Exchange (ETDEWEB)

    Raballand, V; Benedikt, J; Keudell, A von [Research Group Reactive Plasmas, Ruhr-Universitaet Bochum, 44780 Bochum (Germany); Wunderlich, J [Fraunhofer Institut for Process Engineering and Packaging, Giggenhauser Strasse 35, 85354 Freising (Germany)], E-mail: Achim.vonKeudell@rub.de

    2008-06-07

    The inactivation of spores of Bacillus atrophaeus and of Aspergillus niger using beams of argon ions, of oxygen molecules and of oxygen atoms is studied. Thereby, the conditions occurring in oxygen containing low pressure plasmas are mimicked and fundamental inactivation mechanisms can be revealed. It is shown that the impact of O atoms has no effect on the viability of the spores and that no etching of the spore coat occurs up to an O atom fluence of 3.5 x 10{sup 19} cm{sup -2}. The impact of argon ions with an energy of 200 eV does not cause significant erosion for fluences up to 1.15 x 10{sup 18} cm{sup -2}. However, the combined impact of argon ions and oxygen molecules or atoms causes significant etching of the spores and significant inactivation. This is explained by the process of chemical sputtering, where an ion-induced defect at the surface of the spore reacts with either the incident bi-radical O{sub 2} or with an incident O atom. This leads to the formation of CO, CO{sub 2} and H{sub 2}O and thus to erosion.

  14. 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.

  15. Comparative analysis of the sensitivity of metagenomic sequencing and PCR to detect a biowarfare simulant (Bacillus atrophaeus in soil samples.

    Directory of Open Access Journals (Sweden)

    Delphine Plaire

    Full Text Available To evaluate the sensitivity of high-throughput DNA sequencing for monitoring biowarfare agents in the environment, we analysed soil samples inoculated with different amounts of Bacillus atrophaeus, a surrogate organism for Bacillus anthracis. The soil samples considered were a poorly carbonated soil of the silty sand class, and a highly carbonated soil of the silt class. Control soil samples and soil samples inoculated with 10, 103, or 105 cfu were processed for DNA extraction. About 1% of the DNA extracts was analysed through the sequencing of more than 108 reads. Similar amounts of extracts were also studied for Bacillus atrophaeus DNA content by real-time PCR. We demonstrate that, for both soils, high-throughput sequencing is at least equally sensitive than real-time PCR to detect Bacillus atrophaeus DNA. We conclude that metagenomics allows the detection of less than 10 ppm of DNA from a biowarfare simulant in complex environmental samples.

  16. Comparative analysis of the sensitivity of metagenomic sequencing and PCR to detect a biowarfare simulant (Bacillus atrophaeus) in soil samples.

    Science.gov (United States)

    Plaire, Delphine; Puaud, Simon; Marsolier-Kergoat, Marie-Claude; Elalouf, Jean-Marc

    2017-01-01

    To evaluate the sensitivity of high-throughput DNA sequencing for monitoring biowarfare agents in the environment, we analysed soil samples inoculated with different amounts of Bacillus atrophaeus, a surrogate organism for Bacillus anthracis. The soil samples considered were a poorly carbonated soil of the silty sand class, and a highly carbonated soil of the silt class. Control soil samples and soil samples inoculated with 10, 103, or 105 cfu were processed for DNA extraction. About 1% of the DNA extracts was analysed through the sequencing of more than 108 reads. Similar amounts of extracts were also studied for Bacillus atrophaeus DNA content by real-time PCR. We demonstrate that, for both soils, high-throughput sequencing is at least equally sensitive than real-time PCR to detect Bacillus atrophaeus DNA. We conclude that metagenomics allows the detection of less than 10 ppm of DNA from a biowarfare simulant in complex environmental samples.

  17. Biosurfactant Production by Cultivation of Bacillus atrophaeus ATCC 9372 in Semidefined Glucose/Casein-Based Media

    Science.gov (United States)

    Das Neves, Luiz Carlos Martins; de Oliveira, Kátia Silva; Kobayashi, Márcio Junji; Vessoni Penna, Thereza Christina; Converti, Attilio

    Biosurfactants are proteins with detergent, emulsifier, and antimicrobial actions that have potential application in environmental applications such as the treatment of organic pollutants and oil recovery. Bacillus atrophaeus strains are nonpathogenic and are suitable source of biosurfactants, among which is surfactin. The aim of this work is to establish a culture medium composition able to stimulate biosurfactants production by B. atrophaeus ATCC 9372. Batch cultivations were carried out in a rotary shaker at 150 rpm and 35°C for 24 h on glucose- and/or casein-based semidefined culture media also containing sodium chloride, dibasic sodium phosphate, and soy flour. The addition of 14.0 g/L glucose in a culture medium containing 10.0 g/L of casein resulted in 17 times higher biosurfactant production (B max=635.0 mg/L). Besides, the simultaneous presence of digested casein (10.0 g/L), digested soy flour (3.0 g/L), and glucose (18.0 g/L) in the medium was responsible for a diauxic effect during cell growth. Once the diauxie started, the average biosurfactants concentration was 16.8% less than that observed before this phenomenon. The capability of B. atrophaeus strain to adapt its own metabolism to use several nutrients as energy sources and to preserve high levels of biosurfactants in the medium during the stationary phase is a promising feature for its possible application in biological treatments.

  18. Identification and characterization of a new Bacillus atrophaeus strain B5 as biocontrol agent of postharvest anthracnose disease in soursop (Annona muricata) and avocado (Persea americana).

    Science.gov (United States)

    Guardado-Valdivia, Lizeth; Tovar-Pérez, Erik; Chacón-López, Alejandra; López-García, Ulises; Gutiérrez-Martínez, Porfirio; Stoll, Alexandra; Aguilera, Selene

    2018-05-01

    Anthracnose is a fungal disease caused by Colletotrichum species that is detrimental to numerous fruit, including soursop and avocado. The use of fungicides to maintain the high quality of fruit creates a potential health risk. One alternative to this problem is the biological control, which has been applied successfully during postharvest. The Bacillus species are one of the most studied biological agents against postharvest pathogens because accomplish their biocontrol performance by producing a variety of metabolites. In this study, we evaluated the activity of metabolites contained in the cell free supernatant, obtained from Bacillus strain B5 culture, against micelial growth and spore germination of two virulent strains of C. gloeosporioides isolated from soursop and avocado. On the basis of 16S rDNA gene sequence analysis, this strain was identified as Bacillus atrophaeus. A preventive treatment using cell free supernatant, reduced severity and incidence of anthracnose disease on harvested soursop and avocado fruit. B. atrophaeus strain B5 harbors genes involved in the production of antibiotics such as surfactin, bacillomycin and iturin, which could be contributing to the efficiency of the preventive treatment during postharvest. The antagonistic role of metabolites contained in the cell free supernatant against anthracnose disease, provide a new approach by which to attack this problem and can help reduce the use of chemical pesticides, environmental pollution, leading to the safer fruit preservation. Copyright © 2018 Elsevier GmbH. All rights reserved.

  19. Genomic signatures of strain selection and enhancement in Bacillus atrophaeus var. globigii, a historical biowarfare simulant.

    Directory of Open Access Journals (Sweden)

    Henry S Gibbons

    Full Text Available BACKGROUND: Despite the decades-long use of Bacillus atrophaeus var. globigii (BG as a simulant for biological warfare (BW agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. We characterized a lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (WGS. RESULTS: Archival strains and two "present day" type strains were compared to simulant strains on different laboratory media. Several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. To trace the microevolutionary history of these isolates, we obtained WGS data for several archival and present-day strains and morphotypes. Bacillus-wide phylogenetic analysis identified B. subtilis as the nearest neighbor to B. atrophaeus. The genome of B. atrophaeus is, on average, 86% identical to B. subtilis on the nucleotide level. WGS of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the "military" isolates. Metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of "military" isolates on lactate-containing media, and showed that the "military" strains exhibited a hypersporulating phenotype. CONCLUSIONS: Our analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. Together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation.

  20. Development of a low-cost sterilization biological indicator using Bacillus atrophaeus by solid-state fermentation.

    Science.gov (United States)

    Sella, Sandra Regina B R; Guizelini, Belquis P; Zanello, Pedro H; Vandenberghe, Luciana P S; Ribeiro, Ciro A O; Minozzo, João Carlos; Soccol, Carlos Ricardo

    2012-01-01

    The production of biological indicators involving bacterial sporulation and multi-step downstream processes has been described. The goal of the present work was to use fermented material as the final product in a biological indicator, thereby reducing processing steps and costs. The performance of three different inexpensive supports (vermiculite, sand, and sugarcane bagasse) was assessed by determining Bacillus atrophaeus sporulation during solid-state fermentation and by assessing the direct use of the fermentation products in the subsequent steps of the process. All three supports allowed spore production of between 10(7) and 10(9) CFU g(-1). Sand proved to be the best inert support enabling the direct use of the fermented product due to its easy homogenization, filling properties, and compatibility with recovery medium. Bacterial adhesion to the sand surface was supported by biofilm formation. The resistance to sterilization of the dried fermentation product was evaluated. For dry-heat resistance (160°C), the D value was 6.6 min, and for ethylene oxide resistance (650 mg/L), the D value was 6.5 min. The cost reduction of this process was at least 48%. No previous studies have been published on the application of sand as a support in solid-state fermentation for the production of biological indicators.

  1. Bacillus subtilis Spore Inner Membrane Proteome

    NARCIS (Netherlands)

    Zheng, L.; Abhyankar, W.; Ouwerling, N.; Dekker, H.L.; van Veen, H.; van der Wel, N.N.; Roseboom, W.; de Koning, L.J.; Brul, S.; de Koster, C.G.

    2016-01-01

    The endospore is the dormant form of Bacillus subtilis and many other Firmicutes. By sporulation, these spore formers can survive very harsh physical and chemical conditions. Yet, they need to go through germination to return to their growing form. The spore inner membrane (IM) has been shown to

  2. 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.

  3. Sterilization of Bacillus atrophaeus using OH radicals supplied by vacuum ultraviolet method

    Science.gov (United States)

    Yonetamari, Kenta; Tokumitsu, Yusuke; Yonemori, Seiya; Ono, Ryo; Yasuda, Hachiro; Mizuno, Akira

    2015-09-01

    Sterilization by cold plasma has widely been performed. It is well known that reactive oxygen species (ROS) has a potential of sterilization. However, it is not clear which ROS is effective on sterilization because a lot of types of ROS are produced in plasma. In this study, sterilization effect of OH radicals by vacuum ultraviolet (VUV) method was investigated. This method utilizes photodissociation reaction to produce ROS so it can produce ROS selectively. Wet and dry helium with and without 1% O2 gas was used to demonstrate sterilization effect of OH radicals. Gases were flowed in a quartz tube (inner diameter 2 mm, outer diameter 4 mm) at a flow rate of 1.5 L/min. The produced ROS flowed out of the quartz tube nozzle. A Xe2 excimer lamp emitting 172 +/- 7 nm VUV light was placed parallel to the quartz tube with a distance of 8 mm. The distance between the lower end of the lamp and the nozzle of quartz tube was changed from 3 to 15 cm. As a target of sterilization, Bacillus atrophaeus (ATCC 9372) was used. The density of OH radicals was measured using laser-induced fluorescence (LIF). As a result, sterilization using VUV method was verified. This result showed that OH radicals sterilized the bacteria.

  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. 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

  6. Production of lipopeptide biosurfactants by Bacillus atrophaeus 5-2a and their potential use in microbial enhanced oil recovery.

    Science.gov (United States)

    Zhang, Junhui; Xue, Quanhong; Gao, Hui; Lai, Hangxian; Wang, Ping

    2016-10-03

    Lipopeptides are known as promising microbial surfactants and have been successfully used in enhancing oil recovery in extreme environmental conditions. A biosurfactant-producing strain, Bacillus atrophaeus 5-2a, was recently isolated from an oil-contaminated soil in the Ansai oilfield, Northwest China. In this study, we evaluated the crude oil removal efficiency of lipopeptide biosurfactants produced by B. atrophaeus 5-2a and their feasibility for use in microbial enhanced oil recovery. The production of biosurfactants by B. atrophaeus 5-2a was tested in culture media containing eight carbon sources and nitrogen sources. The production of a crude biosurfactant was 0.77 g L -1 and its surface tension was 26.52 ± 0.057 mN m -1 in a basal medium containing brown sugar (carbon source) and urea (nitrogen source). The biosurfactants produced by the strain 5-2a demonstrated excellent oil spreading activity and created a stable emulsion with paraffin oil. The stability of the biosurfactants was assessed under a wide range of environmental conditions, including temperature (up to 120 °C), pH (2-13), and salinity (0-50 %, w/v). The biosurfactants were found to retain surface-active properties under the extreme conditions. Additionally, the biosurfactants were successful in a test to simulate microbial enhanced oil recovery, removing 90.0 and 93.9 % of crude oil adsorbed on sand and filter paper, respectively. Fourier transform infrared spectroscopy showed that the biosurfactants were a mixture of lipopeptides, which are powerful biosurfactants commonly produced by Bacillus species. The study highlights the usefulness of optimization of carbon and nitrogen sources and their effects on the biosurfactants production and further emphasizes on the potential of lipopeptide biosurfactants produced by B. atrophaeus 5-2a for crude oil removal. The favorable properties of the lipopeptide biosurfactants make them good candidates for application in the bioremediation of oil

  7. 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...

  8. Infrared Signatures to Discriminate Viability of Autoclaved Bacillus Spores

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, Matthew D.; Valentine, Nancy B.; Johnson, Timothy J.

    2011-10-06

    Optical methods can offer good sensitivity for detecting small amounts of chemicals and biologicals, and as these methods mature, are some of the few techniques that can offer true standoff detection. For detection of biological species, determining the viability is clearly important: Certain species of gram-positive bacteria are capable of forming endospores, specialized structures that arise when living conditions become unfavorable or little growth medium is available, being resistant to many chemicals as well as changes in heat or pH. Such spores can remain dormant from months to years until more favorable conditions arise, resulting in germination back to the vegetative state. This persistence characteristic of bacterial spores allows for contamination of a surface (e.g. food or medical equipment) even after the surface has been nominally cleaned. Bacterial spores have also been used as biological weapons, as in the case with B. anthracis. Thus, rapid analysis to determine a spore's viability in a given environment or after attempts to sterilize a given environment is crucial. The increasing availability of portable spectrometers may provide a key to such rapid onsite analysis. The present study was designed to determine whether infrared spectroscopy may be used to differentiate between viable vs. dead B. subtilis and B. atrophaeus spores. Preliminary results show that the reproducible differences in the IR signatures can be used to identify viable vs. autoclaved (dead) B. subtilis and B. atrophaeus bacterial spores.

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

    National Research Council Canada - National Science Library

    Brittingham, Katherine C; Ruthel, Gordon; Panchal, Rekha G; Fuller, Claudette L; Ribot, Wilson J

    2005-01-01

    Phagocytosis of inhaled Bacillus anthracis spores and subsequent trafficking to lymph nodes are decisive events in the progression of inhaled anthrax because they initiate germination and dissemination of spores...

  10. Modeling Radiation Effectiveness for Inactivation of Bacillus Spores

    Science.gov (United States)

    2015-09-17

    MODELING RADIATION EFFECTIVENESS FOR INACTIVATION OF BACILLUS SPORES DISSERTATION Emily A. Knight, Major, USAF AFIT-ENC-DS-15-S-001 DEPARTMENT OF THE...not subject to copyright protection in the United States. AFIT-ENC-DS-15-S-001 MODELING RADIATION EFFECTIVENESS FOR INACTIVATION OF BACILLUS SPORES...EFFECTIVENESS FOR INACTIVATION OF BACILLUS SPORES Emily A. Knight, B.A., M.S. Major, USAF Committee Membership: Dr. William P. Baker Chair Dr. Larry W

  11. Inactivation of Bacillus Anthracis Spores Using Carbon Nanotubes

    Science.gov (United States)

    2014-10-30

    effect of SWNTs in combination with antimicrobial chemicals on inactivation of B. anthracis spores; 4) the effect of CNTs coated surfaces on the...2010 31-May-2014 Approved for Public Release; Distribution Unlimited Final Report: (Life Science Division/ Biochemistry ) Inactivation of Bacillus... Biochemistry ) Inactivation of Bacillus Anthracis Spores Using Carbon Nanotubes Report Title The Specific Aims of the project were to investigate: 1) the

  12. Role of DNA repair in Bacillus subtilis spore resistance.

    OpenAIRE

    Setlow, B; Setlow, P

    1996-01-01

    Wet-heat or hydrogen peroxide treatment of wild-type Bacillus subtilis spores did not result in induction of lacZ fusions to three DNA repair-related genes (dinR, recA, and uvrC) during spore outgrowth. However, these genes were induced during outgrowth of wild-type spores treated with dry heat or UV. Wet-heat, desiccation, dry-heat, or UV treatment of spores lacking major DNA-binding proteins (termed alpha-beta- spores) also resulted in induction of the three DNA repair genes during spore ou...

  13. Infrared signatures to discriminate viability of autoclaved Bacillus spores

    Science.gov (United States)

    Schneider, Matthew D. W.; Valentine, Nancy B.; Johnson, Timothy J.

    2011-11-01

    Optical methods can offer good sensitivity for detecting small amounts of chemicals and biologicals, and as these methods mature, are some of the few techniques that can offer true standoff detection. For detection of biological species, determining the viability is clearly important: Certain species of gram-positive bacteria are capable of forming endospores, specialized structures that arise when living conditions become unfavorable or little growth medium is available. Spores are also resistant to many chemicals as well as changes in heat or pH; such spores can remain dormant from months to years until more favorable conditions arise, resulting in germination back to the vegetative state. This persistence characteristic of bacterial spores allows for contamination of a surface (e.g. food or medical equipment) even after the surface has been nominally cleaned. Bacterial spores have also been used as biological weapons, as in the case of B. anthracis. Thus, having rapid analytical methods to determine a spore's viability after attempts to clean a given environment is crucial. The increasing availability of portable spectrometers may provide a key to such rapid onsite analysis. The present study was designed to determine whether infrared spectroscopy may be used to differentiate between viable vs. dead B. subtilis and B. atrophaeus spores. Preliminary results show that the reproducible differences in the IR signatures can be used to identify the viable vs. the autoclaved (dead) spores.

  14. Effect of Ultrasonic Waves on the Heat Resistance of Bacillus cereus and Bacillus licheniformis Spores

    Science.gov (United States)

    Burgos, J.; Ordóñez, J. A.; Sala, F.

    1972-01-01

    Heat resistance of Bacillus cereus and Bacillus licheniformis spores in quarter-strength Ringer solution decreases markedly after ultrasonic treatments which are unable to kill a significant proportion of the spore population. This effect does not seem to be caused by a loss of Ca2+ or dipicolinic acid. The use of ultrasonics to eliminate vegetative cells or to break aggregates in Bacillus spore suspensions to be used subsequently in heat resistance experiments appears to be unadvisable. PMID:4627969

  15. Germination Requirements of Bacillus macerans Spores

    Science.gov (United States)

    Sacks, L. E.; Thompson, P. A.

    1971-01-01

    2-Phenylacetamide is an effective germinant for spores of five strains of Bacillus macerans, particularly in the presence of fructose. Benzyl penicillin, the phenyl acetamide derivative of penicillin, and phenylacetic acid are also good germinants. l-Asparagine is an excellent germinant for four strains. α-Amino-butyric acid is moderately effective. Pyridoxine, pyridoxal, adenine, and 2,6-diaminopurine are potent germinants for NCA strain 7X1 only. d-Glucose is a powerful germinant for strain B-70 only. d-Fructose and d-ribose strongly potentiate germination induced by other germinants (except l-asparagine) but have only weak activity by themselves. Niacinamide and nicotinamide-adenine dinucleotide, inactive by themselves, are active in the presence of fructose or ribose. Effects of pH, ion concentration, and temperature are described. PMID:4251279

  16. Analysis of Bacillus Globigii Spores Using the BioDetector

    National Research Council Canada - National Science Library

    Lee, William

    1999-01-01

    .... An automated immunoassay instrument capable of providing rapid identification of biological agents was used to analyses laboratory and field trial samples containing the field trial simulants Bacillus globigii (BG) spores...

  17. Lipopeptides, a novel protein, and volatile compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus CAB-1.

    Science.gov (United States)

    Zhang, Xiaoyun; Li, Baoqing; Wang, Ye; Guo, Qinggang; Lu, Xiuyun; Li, Shezeng; Ma, Ping

    2013-11-01

    Bacillus atrophaeus CAB-1 displays a high inhibitory activity against various fungal pathogens and suppresses cucumber powdery mildew and tomato gray mold. We extracted and identified lipopeptides and secreted proteins and volatile compounds produced by strain CAB-1 to investigate the mechanisms involved in its biocontrol performance. In vitro assays indicated all three types of products contributed to the antagonistic activity against the fungal pathogen Botrytis cinerea. Each of these components also effectively prevented the occurrence of the cucumber powdery mildew caused by Sphaerotheca fuliginea under greenhouse conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry revealed that the major bioactive lipopeptide was fengycin A (C15-C17). We isolated the crude-secreted proteins of CAB-1 and purified a fraction with antifungal activity. This protein sequence shared a high identity with a putative phage-related pre-neck appendage protein, which has not been reported as an antifungal factor. The volatile compounds produced by CAB-1 were complex, including a range of alcohols, phenols, amines, and alkane amides. O-anisaldehyde represented one of the most abundant volatiles with the highest inhibition on the mycelial growth of B. cinerea. To our knowledge, this is the first report on profiling three types of antifungal substances in Bacilli and demonstrating their contributions to plant disease control.

  18. Germination of Bacillus cereus spores adhered to stainless steel

    NARCIS (Netherlands)

    Hornstra, L.M.; Leeuw, de P.P.L.A.; Moezelaar, R.; Wolbert, E.J.H.; Vries, de Y.P.; Vos, de W.M.; Abee, T.

    2007-01-01

    Adhered spores of Bacillus cereus represent a significant part of the surface-derived contamination in processing equipment used in the dairy industry. As germinated spores lose their resistance capacities instantaneously, efficient germination prior to a cleaning in place treatment could aid to the

  19. 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

  20. Architecture and Assembly of the Bacillus subtilis Spore Coat

    Science.gov (United States)

    2014-09-26

    483 489. 15. Abhyankar W, Ter Beek A, Dekker H, Kort R, Brul S, et al. (2011) Gel-free proteomic identification of the Bacillus subtilis insoluble coat... identification of additional sporulation genes in Bacillus subtilis. J Mol Biol 327: 945 972. AFM of Spore Coat Architecture PLOS ONE | www.plosone.org 16 September 2014 | Volume 9 | Issue 9 | e108560 ...1ITLE AND SUBTITLE 5a CONTRACTNUMBER Architecture and assembly of the Bacillus subtilis spore coat W911NF-09-l-0286 5b. GRANT NUMBER 5c. PROGRAM

  1. 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...

  2. Rapid filtration separation-based sample preparation method for Bacillus spores in powdery and environmental matrices.

    Science.gov (United States)

    Isabel, Sandra; Boissinot, Maurice; Charlebois, Isabelle; Fauvel, Chantal M; Shi, Lu-E; Lévesque, Julie-Christine; Paquin, Amélie T; Bastien, Martine; Stewart, Gale; Leblanc, Eric; Sato, Sachiko; Bergeron, Michel G

    2012-03-01

    Authorities frequently need to analyze suspicious powders and other samples for biothreat agents in order to assess environmental safety. Numerous nucleic acid detection technologies have been developed to detect and identify biowarfare agents in a timely fashion. The extraction of microbial nucleic acids from a wide variety of powdery and environmental samples to obtain a quality level adequate for these technologies still remains a technical challenge. We aimed to develop a rapid and versatile method of separating bacteria from these samples and then extracting their microbial DNA. Bacillus atrophaeus subsp. globigii was used as a simulant of Bacillus anthracis. We studied the effects of a broad variety of powdery and environmental samples on PCR detection and the steps required to alleviate their interference. With a benchmark DNA extraction procedure, 17 of the 23 samples investigated interfered with bacterial lysis and/or PCR-based detection. Therefore, we developed the dual-filter method for applied recovery of microbial particles from environmental and powdery samples (DARE). The DARE procedure allows the separation of bacteria from contaminating matrices that interfere with PCR detection. This procedure required only 2 min, while the DNA extraction process lasted 7 min, for a total of sample preparation procedure allowed the recovery of cleaned bacterial spores and relieved detection interference caused by a wide variety of samples. Our procedure was easily completed in a laboratory facility and is amenable to field application and automation.

  3. 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...display a currently valid OMB control number. 1. REPORT DATE 02 OCT 2015 2. REPORT TYPE N/A 3. DATES COVERED 4. TITLE AND SUBTITLE Inhibiting...inosine hydrolase and alanine racemase to enhance the germination of Bacillus anthracis Sterne spores potential spore decontamination strategies 5a

  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. Architecture and Assembly of the Bacillus subtilis Spore Coat

    Science.gov (United States)

    Plomp, Marco; Carroll, Alicia Monroe; Setlow, Peter; Malkin, Alexander J.

    2014-01-01

    Bacillus spores are encased in a multilayer, proteinaceous self-assembled coat structure that assists in protecting the bacterial genome from stresses and consists of at least 70 proteins. The elucidation of Bacillus spore coat assembly, architecture, and function is critical to determining mechanisms of spore pathogenesis, environmental resistance, immune response, and physicochemical properties. Recently, genetic, biochemical and microscopy methods have provided new insight into spore coat architecture, assembly, structure and function. However, detailed spore coat architecture and assembly, comprehensive understanding of the proteomic composition of coat layers, and specific roles of coat proteins in coat assembly and their precise localization within the coat remain in question. In this study, atomic force microscopy was used to probe the coat structure of Bacillus subtilis wild type and cotA, cotB, safA, cotH, cotO, cotE, gerE, and cotE gerE spores. This approach provided high-resolution visualization of the various spore coat structures, new insight into the function of specific coat proteins, and enabled the development of a detailed model of spore coat architecture. This model is consistent with a recently reported four-layer coat assembly and further adds several coat layers not reported previously. The coat is organized starting from the outside into an outermost amorphous (crust) layer, a rodlet layer, a honeycomb layer, a fibrous layer, a layer of “nanodot” particles, a multilayer assembly, and finally the undercoat/basement layer. We propose that the assembly of the previously unreported fibrous layer, which we link to the darkly stained outer coat seen by electron microscopy, and the nanodot layer are cotH- and cotE- dependent and cotE-specific respectively. We further propose that the inner coat multilayer structure is crystalline with its apparent two-dimensional (2D) nuclei being the first example of a non-mineral 2D nucleation crystallization

  6. Architecture and assembly of the Bacillus subtilis spore coat.

    Science.gov (United States)

    Plomp, Marco; Carroll, Alicia Monroe; Setlow, Peter; Malkin, Alexander J

    2014-01-01

    Bacillus spores are encased in a multilayer, proteinaceous self-assembled coat structure that assists in protecting the bacterial genome from stresses and consists of at least 70 proteins. The elucidation of Bacillus spore coat assembly, architecture, and function is critical to determining mechanisms of spore pathogenesis, environmental resistance, immune response, and physicochemical properties. Recently, genetic, biochemical and microscopy methods have provided new insight into spore coat architecture, assembly, structure and function. However, detailed spore coat architecture and assembly, comprehensive understanding of the proteomic composition of coat layers, and specific roles of coat proteins in coat assembly and their precise localization within the coat remain in question. In this study, atomic force microscopy was used to probe the coat structure of Bacillus subtilis wild type and cotA, cotB, safA, cotH, cotO, cotE, gerE, and cotE gerE spores. This approach provided high-resolution visualization of the various spore coat structures, new insight into the function of specific coat proteins, and enabled the development of a detailed model of spore coat architecture. This model is consistent with a recently reported four-layer coat assembly and further adds several coat layers not reported previously. The coat is organized starting from the outside into an outermost amorphous (crust) layer, a rodlet layer, a honeycomb layer, a fibrous layer, a layer of "nanodot" particles, a multilayer assembly, and finally the undercoat/basement layer. We propose that the assembly of the previously unreported fibrous layer, which we link to the darkly stained outer coat seen by electron microscopy, and the nanodot layer are cotH- and cotE- dependent and cotE-specific respectively. We further propose that the inner coat multilayer structure is crystalline with its apparent two-dimensional (2D) nuclei being the first example of a non-mineral 2D nucleation crystallization

  7. Decontamination of Bacillus spores adhered to iron and ...

    Science.gov (United States)

    Journal Article This study examines the effectiveness of decontaminating Bacillus globigii spores attached to corroded iron and cement-mortar coupons with free chlorine at two pH levels, monochloramine, chlorine dioxide, ozone, peracetic acid (PAA) and acidified nitrite, followed by flushing.

  8. DNA fingerprinting of spore-forming bacterial isolates, using Bacillus ...

    African Journals Online (AJOL)

    User

    Bc-repetitive extragenic palindromic polymerase chain reaction (Bc-Rep PCR) analysis was conducted on seven Bacillus thuringiensis isolates accessed from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) culture collection and on five local isolates of entomopathogenic spore- forming bacteria.

  9. Effects of Ingesting Bacillus Thuringiensis (Berliner) Spores on ...

    African Journals Online (AJOL)

    Bacillus thuringiensis Berliner was isolated from dead Sesamia calamistis Hampson (Lepidoptera: Noctuidae) larvae collected from maize farms in Cape Coast, Ghana. Spores produced from the vegetative cells were incorporated into an artificial diet and fed to 2nd instar S. calamistis larvae. The duration of larval and pupal ...

  10. Effects of Ingesting Bacillus Thuringiensis (Berliner) Spores on ...

    African Journals Online (AJOL)

    Effects of Ingesting Bacillus Thuringiensis (Berliner) Spores on Developmental Stages and Fecundity of Surviving Sesamia Calamistis (Hampson) (Lepidoptera: ... The PDF file you selected should load here if your Web browser has a PDF reader plug-in installed (for example, a recent version of Adobe Acrobat Reader).

  11. DNA fingerprinting of spore-forming bacterial isolates, using Bacillus ...

    African Journals Online (AJOL)

    Bc-repetitive extragenic palindromic polymerase chain reaction (Bc-Rep PCR) analysis was conducted on seven Bacillus thuringiensis isolates accessed from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) culture collection and on five local isolates of entomopathogenic spore-forming bacteria.

  12. 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

    International Nuclear Information System (INIS)

    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

  13. Bacillus cereus spore formation, structure and germination

    NARCIS (Netherlands)

    Vries, de Y.P.

    2006-01-01

    Bacterial spores arespecializeddifferentiated celltypes for

  14. Tip-enhanced Raman scattering of bacillus subtilis spores

    Science.gov (United States)

    Rusciano, G.; Zito, G.; Pesce, G.; Sasso, A.; Isticato, R.; Ricca, E.

    2015-07-01

    Understanding of the complex interactions of molecules at biological interfaces is a fundamental issue in biochemistry, biotechnology as well as biomedicine. A plethora of biological processes are ruled by the molecular texture of cellular membrane: cellular communications, drug transportations and cellular recognition are just a few examples of such chemically-mediated processes. Tip-Enhanced Raman Scattering (TERS) is a novel, Raman-based technique which is ideally suited for this purpose. TERS relies on the combination of scanning probe microscopy and Raman spectroscopy. The basic idea is the use of a metalled tip as a sort of optical nano-antenna, which gives place to SERS effect close to the tip end. Herein, we present the application of TERS to analyze the surface of Bacillus subtilis spores. The choice of this biological systems is related to the fact that a number of reasons support the use of spores as a mucosal delivery system. The remarkable and well-documented resistance of spores to various environmental and toxic effects make them clear potentials as a novel, surface-display system. Our experimental outcomes demonstrate that TERS is able to provide a nano-scale chemical imaging of spore surface. Moreover, we demonstrate that TERS allows differentiation between wilde-type spore and genetically modified strains. These results hold promise for the characterization and optimization of spore surface for drug-delivery applications.

  15. 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

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

    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

  17. 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

  18. 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. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  19. Characterization of spore laccase from Bacillus subtilis WD23 and ...

    African Journals Online (AJOL)

    The strain was identified as Bacillus subtilis based on its morphological and physiological properties, and 16S rDNA sequence analysis. The optimum pH and temperature for the spore-bound laccase were 6.8 and 60°C, respectively. The temperature half-life of the laccase was 2.5 h at 80°C and 68 h at 60°C. It also showed ...

  20. Mutagenic effect of tritated water on spores of Bacillus subtilis

    International Nuclear Information System (INIS)

    Tanooka, H.; Munakata, N.

    1978-01-01

    The mutagenic effect of tritiated water was observed with spores of Bacillus subtilis polA strain suspended in 50 mCi/ml of tritiated water for various intervals. Dose rate given by tritium beta particles to spore core was estimated to be 400 rad/hr from some assumptions and E. coli data computed by Bockrath et al. and Sands et al. The initial mutation rate was 4.2 x 10 -9 mutants/rad, as compared with 2.4 x 10 -9 mutants/rad for 60 Co γ rays and 3.3 x 10 -9 mutants/rad for 30-kVp x rays. The mutagenic effect of tritiated water on spores is most likely due to beta particle ionizing radiation damage

  1. Thermal inactivation kinetics of Bacillus coagulans spores in tomato juice.

    Science.gov (United States)

    Peng, Jing; Mah, Jae-Hyung; Somavat, Romel; Mohamed, Hussein; Sastry, Sudhir; Tang, Juming

    2012-07-01

    The thermal characteristics of the spores and vegetative cells of three strains of Bacillus coagulans (ATCC 8038, ATCC 7050, and 185A) in tomato juice were evaluated. B. coagulans ATCC 8038 was chosen as the target microorganism for thermal processing of tomato products due to its spores having the highest thermal resistance among the three strains. The thermal inactivation kinetics of B. coagulans ATCC 8038 spores in tomato juice between 95 and 115°C were determined independently in two different laboratories using two different heating setups. The results obtained from both laboratories were in general agreement, with z-values (z-value is defined as the change in temperature required for a 10-fold reduction of the D-value, which is defined as the time required at a certain temperature for a 1-log reduction of the target microorganisms) of 8.3 and 8.7°C, respectively. The z-value of B. coagulans 185A spores in tomato juice (pH 4.3) was found to be 10.2°C. The influence of environmental factors, including cold storage time, pH, and preconditioning, upon the thermal resistance of these bacterial spores is discussed. The results obtained showed that a storage temperature of 4°C was appropriate for maintaining the viability and thermal resistance of B. coagulans ATCC 8038 spores. Acidifying the pH of tomato juice decreased the thermal resistance of these spores. A 1-h exposure at room temperature was considered optimal for preconditioning B. coagulans ATCC 8038 spores in tomato juice.

  2. A Novel Spectroscopic Methodology for the Investigation of Individual Bacillus Spores

    National Research Council Canada - National Science Library

    Alexander, Troy A; Pellegrino, Paul; Gillespie, James B

    2005-01-01

    A methodology has been developed for the investigation of bacterial spores. Specifically, this method has been used to probe the spore coat composition of two different Bacillus stearothermophilus variants...

  3. Detection of Bacillus spores using PCR and FTA filters.

    Science.gov (United States)

    Lampel, Keith A; Dyer, Deanne; Kornegay, Leroy; Orlandi, Palmer A

    2004-05-01

    Emphasis has been placed on developing and implementing rapid detection systems for microbial pathogens. We have explored the utility of expanding FTA filter technology for the preparation of template DNA for PCR from bacterial spores. Isolated spores from several Bacillus spp., B. subtilis, B. cereus, and B. megaterium, were applied to FTA filters, and specific DNA products were amplified by PCR. Spore preparations were examined microscopically to ensure that the presence of vegetative cells, if any, did not yield misleading results. PCR primers SRM86 and SRM87 targeted a conserved region of bacterial rRNA genes, whereas primers Bsub5F and Bsub3R amplified a product from a conserved sequence of the B. subtilis rRNA gene. With the use of the latter set of primers for nested PCR, the sensitivity of the PCR-based assay was increased. Overall, 53 spores could be detected after the first round of PCR, and the sensitivity was increased to five spores by nested PCR. FTA filters are an excellent platform to remove PCR inhibitors and have universal applications for environmental, clinical, and food samples.

  4. Germination of Bacillus cereus spores adhered to stainless steel.

    Science.gov (United States)

    Hornstra, L M; de Leeuw, P L A; Moezelaar, R; Wolbert, E J; de Vries, Y P; de Vos, W M; Abee, T

    2007-05-30

    Adhered spores of Bacillus cereus represent a significant part of the surface-derived contamination in processing equipment used in the dairy industry. As germinated spores lose their resistance capacities instantaneously, efficient germination prior to a cleaning in place treatment could aid to the disinfecting effect of such a treatment. Therefore, spores of B. cereus ATCC 14579 and that of the environmental isolate B. cereus CMCC 3328 were assessed for their germination behaviour when adhered to a stainless steel surface. A mixture of l-alanine and inosine initiated germination of adhered spores efficiently, resulting in 3.2 decimal logarithms of germination. Notably, implementation of a germination-inducing step prior to a representative cleaning in place procedure reduced the number of survivors with over 3 decimal log units, while an alkali treatment alone, as part of the cleaning in place procedure, did not show any effect on B. cereus spore viability. These results show that implementation of a germination step enhances the disinfection effect of currently used cleaning in place procedures.

  5. Characterization of the Surface Morphology of Bacillus Spores by Atomic Force Microscopy

    National Research Council Canada - National Science Library

    Zolock, Ruth

    2002-01-01

    .... kurstaki, Bacillus cereus strain 569, and Bacillus globigii var. niger. The spores were separated from a nutrient agar culture by filtering and centrifugation, suspended in deionized water, and immobilized on a graphite substrate by spin-coating...

  6. 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.

  7. Decontamination of Bacillus subtilis Spores in a Sealed Package Using a Non-thermal Plasma System

    Science.gov (United States)

    Keener, Kevin M.; Jensen, J. L.; Valdramidis, V. P.; Byrne, E.; Connolly, J.; Mosnier, J. P.; Cullen, P. J.

    The safety of packaged food and medical devices is a major concern to consumers and government officials. Recent inventions (PK-1 and PK-2) based on the principles of non-thermal, atmospheric plasma has shown significant reduction in bacterial contamination inside a sealed package. The objective of this study was to evaluate the PK-1 and PK-2 systems in the reduction of Bacillus subtilis spores using packages containing air or modified atmosphere (MA) gas (65% O2/30% CO2/5% N2). The experimental design consisted of the following parameters: (1) two voltage conditions: 13.5 kV with 1.0 cm electrode gap (PK-1) and 80 kV with 4.5 cm electrode gap (PK-2), (2) two treatment conditions: inside and outside the field of ionization, (3) PK-1 and PK-2 optimized treatment times: 300 and 120 s, respectively, and (4) two package gas types: air and modified atmosphere (MA) gas (65% O2/30% CO2/5% N2). Measurements included: (1) bacterial reductions of Bacillus subtilis var. niger (B. atrophaeus), (2) ozone, nitrous oxides (NOx), and carbon monoxide concentrations, and (3) relative humidity. Bacillus subtilis (1.7 × 106/strip) were loaded into sterile uncovered petri dishes and treated with ionization generated in packages using air or MA gas blend. Samples were treated for 300 s (PK-1) or 120 s (PK-2) and stored at room ­temperature for 24 h. Results documented relative humidity (RH) ranged from 20% to 30%. After 300 s of PK-1 treatment (13.5 kV/44 W/1.0 cm gap), ozone concentrations were 6,000 ppm (air) and 7,500 ppm (MA). After 120 s of PK-2 treatment (80 kV/150 W/4.5 cm), ozone concentrations were 7,500 ppm (air) and 12,000 ppm (MA). Ozone and NOx concentrations were non-detect (ND) after 24 h. PK-1 carbon monoxide levels were package ionization process.

  8. Induction of prophages in spores of Bacillus subtilis by ultraviolet irradiation from synchrotron orbital radiation

    Energy Technology Data Exchange (ETDEWEB)

    Sadaie, Y.; Kada, T.; Ohta, Y. (National Inst. of Genetics, Mishima, Shizuoka (Japan)); Kobayashi, K.; Hieda, K.; Ito, T.

    1984-06-01

    Prophages were induced from Bacillus subtilis spores lysogenic with SP02 by ultraviolet (160 nm to 240 nm) irradiation from synchrotron orbital radiation (SR UV). SR UV at around 220 nm was most effective in the inactivation of spores and prophage induction from lysogenic spores, suggesting that the lesions are produced on the DNA molecule which eventually induces signals to inactivate the phage repressor.

  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. 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

    13/2011 22.00 Keren K. Griffiths, Jingqiao Zhang, Ann E. Cowan, Ji Yu, Peter Setlow. Germination proteins in the inner membrane of dormant Bacillus...that this technique can be used to rapidly identify single airborne particles or bacteria collected on a slide and to monitor germination dynamics of...the environment of dipicolinic acid in the core of superdormant spores is different from that in dormant spores [J. Bacteriol., 191, 5584 (2009

  12. Lethality of Bacillus Anthracis Spores Due to Short Duration Heating Measured Using Infrared Spectroscopy

    National Research Council Canada - National Science Library

    Goetz, Kristina M

    2005-01-01

    In this research, Bacillus anthracis spores were subjected to bursts of heat lasting on the order of one second in duration using a laser system to simulate the explosive environment from an agent defeat weapon...

  13. Characterization of the Surface Morphology of Bacillus Spores by Atomic Force Microscopy

    National Research Council Canada - National Science Library

    Zolock, Ruth

    2002-01-01

    The surface morphology of Bacillus spores was resolved by atomic force microscopy in order to determine if characteristic surface features could be used to distinguish between closely related species...

  14. The search and identification of the new immunodiagnostic targets of bacillus anthracis spore

    International Nuclear Information System (INIS)

    Biketov, S.; Dunaytsev, I.; Baranova, E.; Marinin, L.; Dyatlov, I.

    2009-01-01

    Spores of Bacillus anthracis have been used as bio warfare agent to bio terrorize purposes. As efficiency of anti-epidemic measures included urgent prevention and treatment is determined by terms within which the bio agent is identified. Direct and rapid spore detection by antibodies based detection system is very attractive alternative to current PCR-based assays or routine phenotyping which are the most accurate but are also complex, time-consumption and expensive. The main difficulty with respect to such kind of anthrax spores detection is a cross-reaction with spores of closely related bacteria. For development of species-specific antibodies to anthrax spores recombinant scFvs or hybridoma technique were used. In both case surface spore antigens contained species-specific epitopes are need. Among exosporium proteins only ExsF(BxpB), ExsK and SoaA are specific to B.cereus group. On the surface of B. anthracis spores, a unique tetrasaccharides containing an novel monosaccharide - anthrose, was discovered. It was shown that anthrose can be serving as species-specific target for B. anthracis spores detection. We have revealed that EA1 isolated from spore of Russians strain STI-1 contain carbohydrate which formed species-specific epitopes and determine immunogenicity of this antigen. Antibodies to this antigen specifically recognized the surface target of B. anthracis spores and do not reacted with others Bacillus spore. Based on these antibodies we developed the test-systems in different formats for rapid direct detection and identification of B. anthracis spores. The results of trial these test-systems with using more than 50 different Bacillus strains were indicated that carbohydrate of EA1 isolated from spore is effective immunodiagnostic target for anthrax spores bio detection.(author)

  15. 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 ...

  16. A probabilistic modeling approach in thermal inactivation: estimation of postprocess Bacillus cereus spore prevalence and concentration

    NARCIS (Netherlands)

    Membre, J.M.; Amezquita, A.; Bassett, J.; Giavedoni, P.; Blackburn, W.; Gorris, L.G.M.

    2006-01-01

    The survival of spore-forming bacteria is linked to the safety and stability of refrigerated processed foods of extended durability (REPFEDs). A probabilistic modeling approach was used to assess the prevalence and concentration of Bacillus cereus spores surviving heat treatment for a semiliquid

  17. Improvement of Biological Indicators by Uniformly Distributing Bacillus subtilis Spores in Monolayers To Evaluate Enhanced Spore Decontamination Technologies.

    Science.gov (United States)

    Raguse, Marina; Fiebrandt, Marcel; Stapelmann, Katharina; Madela, Kazimierz; Laue, Michael; Lackmann, Jan-Wilm; Thwaite, Joanne E; Setlow, Peter; Awakowicz, Peter; Moeller, Ralf

    2016-01-22

    Novel decontamination technologies, including cold low-pressure plasma and blue light (400 nm), are promising alternatives to conventional surface decontamination methods. However, the standardization of the assessment of such sterilization processes remains to be accomplished. Bacterial endospores of the genera Bacillus and Geobacillus are frequently used as biological indicators (BIs) of sterility. Ensuring standardized and reproducible BIs for reliable testing procedures is a significant problem in industrial settings. In this study, an electrically driven spray deposition device was developed, allowing fast, reproducible, and homogeneous preparation of Bacillus subtilis 168 spore monolayers on glass surfaces. A detailed description of the structural design as well as the operating principle of the spraying device is given. The reproducible formation of spore monolayers of up to 5 × 10(7) spores per sample was verified by scanning electron microscopy. Surface inactivation studies revealed that monolayered spores were inactivated by UV-C (254 nm), low-pressure argon plasma (500 W, 10 Pa, 100 standard cubic cm per min), and blue light (400 nm) significantly faster than multilayered spores were. We have thus succeeded in the uniform preparation of reproducible, highly concentrated spore monolayers with the potential to generate BIs for a variety of nonpenetrating surface decontamination techniques. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. Sensitivity of thermally treated Bacillus subtilis spores to subsequent irradiation

    International Nuclear Information System (INIS)

    Mostafa, S.A.; El-Zawahry, Y.A.; Awny, N.M.

    1986-01-01

    B. subtilis spores exposed to thermal treatment at 70 or 80 0 C for 1 hr were more sensitive to subsequent radiation exposure than non-heated spores. Deactivation of previously heated spores by increasing dose of 0-radiation followed an exponential function while, for non-heated spores a shoulder followed by exponential deactivation was noticed. Combined heat-radiation treatment exhibited a synergistic effect on spore deactivation at low irradiation doses, while at high irradiation doses, the effect was more or less additive. Added values of spore injury was higher for B. subtilis spores that received heat and radiation separately than the observed injury for spores that received combined treatment (heat followed by radiation). Results of spore deactivation and injury due to heat followed by radiation treatment are discussed in comparison to those of spores that received radiation-heat sequence

  19. Live cell imaging of germination and outgrowth of individual Bacillus subtilis spores; the effect of heat stress quantitatively analyzed with SporeTracker

    NARCIS (Netherlands)

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

    2013-01-01

    Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more

  20. 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.

  1. 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.

  2. Effects of High Pressure on Bacillus licheniformis Spore Germination and Inactivation.

    Science.gov (United States)

    Borch-Pedersen, Kristina; Mellegård, Hilde; Reineke, Kai; Boysen, Preben; Sevenich, Robert; Lindbäck, Toril; Aspholm, Marina

    2017-07-15

    Bacillus and Clostridium species form spores, which pose a challenge to the food industry due to their ubiquitous nature and extreme resistance. Pressurization at 300 MPa likely triggers germination by opening dipicolinic acid (DPA) channels present in the inner membrane of the spores. In this work, we expose spores of Bacillus licheniformis , a species associated with food spoilage and occasionally with food poisoning, to high pressure (HP) for holding times of up to 2 h. By using mutant spores lacking one or several GRs, we dissect the roles of the GerA, Ynd, and GerK GRs in moderately HP (mHP; 150 MPa)-induced spore germination. We show that Ynd alone is sufficient for efficient mHP-induced spore germination. GerK also triggers germination with mHP, although at a reduced germination rate compared to that of Ynd. GerA stimulates mHP-induced germination but only in the presence of either the intact GerK or Ynd GR. These results suggests that the effectiveness of the individual GRs in mHP-induced germination differs from their effectiveness in nutrient-induced germination, where GerA plays an essential role. In contrast to Bacillus subtilis spores, treatment with very HP (vHP) of 550 MPa at 37°C did not promote effective germination of B. licheniformis spores. However, treatment with vHP in combination with elevated temperatures (60°C) gave a synergistic effect on spore germination and inactivation. Together, these results provide novel insights into how HP affects B. licheniformis spore germination and inactivation and the role of individual GRs in this process. IMPORTANCE Bacterial spores are inherently resistant to food-processing regimes, such as high-temperature short-time pasteurization, and may therefore compromise food durability and safety. The induction of spore germination facilitates subsequent inactivation by gentler processing conditions that maintain the sensory and nutritional qualities of the food. High-pressure (HP) processing is a nonthermal

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

    Science.gov (United States)

    Felker, Daniel L.; Burggraf, Larry W.

    2014-01-01

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

  4. Recent progress in Bacillus subtilis spore-surface display: concept, progress, and future.

    Science.gov (United States)

    Wang, He; Wang, Yunxiang; Yang, Ruijin

    2017-02-01

    With the increased knowledge on spore structure and advances in biotechnology engineering, the newly developed spore-surface display system confers several inherent advantages over other microbial cell-surface display systems including enhanced stability and high safety. Bacillus subtilis is the most commonly used Bacillus species for spore-surface display. The expression of heterologous antigen or protein on the surface of B. subtilis spores has now been practiced for over a decade with noteworthy success. As an update and supplement to other previous reviews, we comprehensively summarize recent studies in the B. subtilis spore-surface display technique. We focus on its benefits as well as the critical factors affecting its display efficiency and offer suggestions for the future success of this field.

  5. Bacillus spores and their relevant chemicals studied by terahertz time domain spectroscopy

    Science.gov (United States)

    Tang, Jianhua; Yang, Bin; Llewellyn, Ian; Cutler, Ronald R.; Donnan, Robert S.

    2014-01-01

    Terahertz time domain spectroscopy has been used to investigate 0.2-2.2 THz transmission responses of Bacillus spores and their related chemical components. Whilst no THz signatures could be clearly associated with either sporulated cells or their chief chemical components, differing degrees of signal attenuation and frequency-dependent light scattering were observed depending on spore composition and culture media. The observed monotonic increase in absorption by spores over this THz spectral domain is mainly from Mie scattering and also from remnant water bound to the spores.

  6. 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

    2010-01-01

    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. PMID:20543054

  7. Germination of Bacillus cereus spores : the role of germination receptors

    NARCIS (Netherlands)

    Hornstra, L.M.

    2007-01-01

    The Bacillus cereus sensu lato group forms a highly homogeneous subdivision of the genus Bacillus and comprises several species that are relevant for humans. Notorious is Bacillus anthracis, the cause of the often-lethal disease anthrax, while the insect pathogen Bacillus

  8. Levels of glycine betaine in growing cells and spores of Bacillus species and lack of effect of glycine betaine on dormant spore resistance.

    Science.gov (United States)

    Loshon, Charles A; Wahome, Paul G; Maciejewski, Mark W; Setlow, Peter

    2006-04-01

    Bacteria of various Bacillus species are able to grow in media with very high osmotic strength in part due to the accumulation of low-molecular-weight osmolytes such as glycine betaine (GB). Cells of Bacillus species grown in rich and minimal media contained low levels of GB, but GB levels were 4- to 60-fold higher in cells grown in media with high salt. GB levels in Bacillus subtilis cells grown in minimal medium were increased approximately 7-fold by GB in the medium and 60-fold by GB plus high salt. GB was present in spores of Bacillus species prepared in media with or without high salt but at lower levels than in comparable growing cells. With spores prepared in media with high salt, GB levels were highest in B. subtilis spores and > or =20-fold lower in B. cereus and B. megaterium spores. Although GB levels in B. subtilis spores were elevated 15- to 30-fold by GB plus high salt in sporulation media, GB levels did not affect spore resistance. GB levels were similar in wild-type B. subtilis spores and spores that lacked major small, acid-soluble spore proteins but were much lower in spores that lacked dipicolinic acid.

  9. Involvement of alanine racemase in germination of Bacillus cereus spores lacking an intact exosporium.

    Science.gov (United States)

    Venir, Elena; Del Torre, Manuela; Cunsolo, Vincenzo; Saletti, Rosaria; Musetti, Rita; Stecchini, Mara Lucia

    2014-02-01

    The L-alanine mediated germination of food isolated Bacillus cereus DSA 1 spores, which lacked an intact exosporium, increased in the presence of D-cycloserine (DCS), which is an alanine racemase (Alr) inhibitor, reflecting the activity of the Alr enzyme, capable of converting L-alanine to the germination inhibitor D-alanine. Proteomic analysis of the alkaline extracts of the spore proteins, which include exosporium and coat proteins, confirmed that Alr was present in the B. cereus DSA 1 spores and matched to that encoded by B. cereus ATCC 14579, whose spore germination was strongly affected by the block of conversion of L- to D-alanine. Unlike ATCC 14579 spores, L-alanine germination of B. cereus DSA 1 spores was not affected by the preincubation with DCS, suggesting a lack of restriction in the reactant accessibility.

  10. 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.

  11. Bacillus amyloliquefaciens Spore Production Under Solid-State Fermentation of Lignocellulosic Residues.

    Science.gov (United States)

    Berikashvili, Violet; Sokhadze, Kakha; Kachlishvili, Eva; Elisashvili, Vladimir; Chikindas, Michael L

    2017-12-16

    This study was conducted to elucidate cultivation conditions determining Bacillus amyloliquefaciens B-1895 growth and enhanced spore formation during the solid-state fermentation (SSF) of agro-industrial lignocellulosic biomasses. Among the tested growth substrates, corncobs provided the highest yield of spores (47 × 10 10 spores g -1 biomass) while the mushroom spent substrate and sunflower oil mill appeared to be poor growth substrates for spore formation. Maximum spore yield (82 × 10 10 spores g -1 biomass) was achieved when 15 g corncobs were moistened with 60 ml of the optimized nutrient medium containing 10 g peptone, 2 g KH 2 PO 4 , 1 g MgSO 4 ·7H 2 O, and 1 g NaCl per 1 l of distilled water. The cheese whey usage for wetting of lignocellulosic substrate instead water promoted spore formation and increased the spore number to 105 × 10 10 spores g -1 . Addition to the cheese whey of optimized medium components favored sporulation process. The feasibility of developed medium and strategy was shown in scaled up SSF of corncobs in polypropylene bags since yield of 10 × 10 11 spores per gram of dry biomass was achieved. In the SSF of lignocellulose, B. amyloliquefaciens B-1895 secreted comparatively high cellulase and xylanase activities to ensure good growth of the bacterial culture.

  12. The Ultraviolet Photochemistry and Photobiology of Vegetative Cells and Spores of Bacillus megaterium

    Science.gov (United States)

    Donnellan, J. E.; Stafford, R. S.

    1968-01-01

    The ultraviolet (UV) photochemistry and photobiology of spores and vegetative cells of Bacillus megaterium have been studied. The response of vegetative cells of B. megaterium appears qualitatively similar to those of Escherichia coli, Micrococcus radiodurans, and Bacillus subtilis with respect to photoproduct formation and repair mechanisms. UV irradiation, however, does not produce cyclobutane-type thymine dimers in the DNA of spores, although other thymine photo-products are produced. The photoproducts do not disappear after photoreactivation, but they are eliminated from the DNA by a dark-repair mechanism different from that found for dimers in vegetative cells. Irradiations performed at three wavelengths produce the same amounts of spore photoproduct and give the same survival curves. Variation of the sporulation medium before irradiation results in comparable alterations in the rate of spore photoproduct production and in survival. PMID:4966691

  13. Mutation induction in spores of Bacillus subtilis by accelerated very heavy ions

    International Nuclear Information System (INIS)

    Baltschukat, K.; Horneck, G.; Buecker, H.; Facius, R.; Schaefer, M.

    1986-01-01

    Mutation induction (resistance to sodium azide) in spores of Bacillus subtilis was investigated after irradiation with heavy ions from Neon to Uranium with specific particle energies between 0.17 and 18.6 MeV/u. A strong dependence of the mutation induction cross section on particle charge and energy was observed. From the results it was concluded that mutation induction in bacterial spores by very heavy ions is mainly caused by secondary electrons. (orig.)

  14. The impact of inducing germination of Bacillus anthracis and Bacillus thuringiensis spores on potential secondary decontamination strategies.

    Science.gov (United States)

    Omotade, T O; Bernhards, R C; Klimko, C P; Matthews, M E; Hill, A J; Hunter, M S; Webster, W M; Bozue, J A; Welkos, S L; Cote, C K

    2014-12-01

    Decontamination and remediation of a site contaminated by the accidental or intentional release of fully virulent Bacillus anthracis spores are difficult, costly and potentially damaging to the environment. Development of novel decontamination strategies that have minimal environmental impacts remains a high priority. Although ungerminated spores are amongst the most resilient organisms known, once exposed to germinants, the germinating spores, in some cases, become susceptible to antimicrobial environments. We evaluated the concept that once germinated, B. anthracis spores would be less hazardous and significantly easier to remediate than ungerminated dormant spores. Through in vitro germination and sensitivity assays, we demonstrated that upon germination, B. anthracis Ames spores and Bacillus thuringiensis Al Hakam spores (serving as a surrogate for B. anthracis) become susceptible to environmental stressors. The majority of these germinated B. anthracis and B. thuringiensis spores were nonviable after exposure to a defined minimal germination-inducing solution for prolonged periods of time. Additionally, we examined the impact of potential secondary disinfectant strategies including bleach, hydrogen peroxide, formaldehyde and artificial UV-A, UV-B and UV-C radiation, employed after a 60-min germination-induction step. Each secondary disinfectant employs a unique mechanism of killing; as a result, germination-induction strategies are better suited for some secondary disinfectants than others. These results provide evidence that the deployment of an optimal combination strategy of germination-induction/secondary disinfection may be a promising aspect of wide-area decontamination following a B. anthracis contamination event. By inducing spores to germinate, our data confirm that the resulting cells exhibit sensitivities that can be leveraged when paired with certain decontamination measures. This increased susceptibility could be exploited to devise more

  15. Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores.

    Science.gov (United States)

    Cho, Eun-Ah; Seo, Jiyoung; Lee, Dong-Woo; Pan, Jae-Gu

    2011-06-10

    Blue multicopper oxidases, laccases displayed on the surface of Bacillus spores were used to decolorize a widely used textile dyestuff, indigo carmine. The laccase-encoding gene of Bacillus subtilis, cotA, was cloned and expressed in B. subtilis DB104, and the expressed enzyme was spontaneously localized on Bacillus spores. B. subtilis spores expressing laccase exhibited maximal activity for the oxidation of 2,2'-azino-bis (3-ethylthiazoline-6-sulfonate) (ABTS) at pH 4.0 and 80°C, and for the decolorization of indigo carmine at pH 8.0 and 60°C. The displayed enzyme retained 80% of its original activity after pre-treatment with organic solvents such as 50% acetonitrile and n-hexane for 2h at 37°C. The apparent K(m) of the enzyme displayed on spores was 443±124 μM for ABTS with a V(max) of 150 ± 16 U/mg spores. Notably, 1mg of spores displaying B. subtilis laccase (3.4 × 10(2)U for ABTS as a substrate) decolorized 44.6 μg indigo carmine in 2h. The spore reactor (0.5 g of spores corresponding to 1.7×10(5)U in 50 mL) in a consecutive batch recycling mode decolorized 223 mg indigo carmine/L to completion within 42 h at pH 8.0 and 60°C. These results suggest that laccase displayed on B. subtilis spores can serve as a powerful environmental tool for the treatment of textile dye effluent. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Heat and desiccation are the predominant factors affecting inactivation of Bacillus licheniformis and Bacillus thuringiensis spores during simulated composting.

    Science.gov (United States)

    Stanford, K; Harvey, A; Barbieri, R; Xu, S; Reuter, T; Amoako, K K; Selinger, L B; McAllister, T A

    2016-01-01

    The suitability of composting for disposal of livestock mortalities due to Bacillus anthracis was assessed by measuring viability of surrogate spores from two strains each of Bacillus licheniformis and Bacillus thuringiensis after a heating cycle modelled on a cattle composting study. Sporulation was attempted from 10 to 37°C, but poor yields at lower temperatures resulted in 25, 30 and 37°C being selected to generate sufficient spores (8 log10  CFU ml(-1) ) for experiments. Spores were inoculated into 3 g autoclaved dried-ground compost rehydrated with 6 ml water or silica beads in a factorial design for each strain, sporulation temperature, matrix and sampling day (0, 25, 50, 100, 150). Maximum incubation temperature was 62°C, but spores were maintained at ≥55°C for 78 of 150 days. Although significant differences existed among Bacillus strains and sporulation temperatures, numbers of viable spores after 150 days averaged 1·3 log10  CFU g(-1) , a 5·2 log10 reduction from day 0. Spore inactivation was likely due to heat and desiccation as matrices were autoclaved prior to incubation, negating impacts of microflora. Results support composting for disposal of anthrax mortalities, provided long-term thermophillic heating is achieved. Due to limited sporulation at 10°C, livestock mortalities from anthrax at this or lower ambient temperatures would likely be of lower risk for disease transmission. © 2015 The Society for Applied Microbiology.

  17. Esterase activity as a novel parameter of spore germination in Bacillus anthracis

    International Nuclear Information System (INIS)

    Ferencko, Linda; Cote, Mindy A.; Rotman, Boris

    2004-01-01

    Spores of Bacillus anthracis were shown to produce esterase activity about 4 min after exposure to conventional germinants such as combinations of amino acids and purine ribosides. Neither amino acids nor ribosides alone induce germination and esterase activity. Expression of esterase activity was chloramphenicol resistant, and correlated with loss of spore refractivity, a traditional parameter of early germination. Based on these observations, we hypothesized that esterase activity could be used as a novel parameter for quantifying early events during spore germination. To test this hypothesis, we measured expression of esterase activity under a variety of germinating conditions. Using diacetyl fluorescein as fluorogenic substrate of esterases, we demonstrated that esterase activity was invariably induced whenever spores were triggered by known germinants. Moreover, D-alanine, an inhibitor of L-alanine-mediated germination, was found to significantly inhibit expression of esterase activity. In terms of molecular mechanisms, esterase expression could represent activation of proteases at the onset of spore germination

  18. Two distinct groups within the Bacillus subtilis group display significantly different spore heat resistance properties.

    Science.gov (United States)

    Berendsen, Erwin M; Zwietering, Marcel H; Kuipers, Oscar P; Wells-Bennik, Marjon H J

    2015-02-01

    The survival of bacterial spores after heat treatment and the subsequent germination and outgrowth in a food product can lead to spoilage of the food product and economical losses. Prediction of time-temperature conditions that lead to sufficient inactivation requires access to detailed spore thermal inactivation kinetics of relevant model strains. In this study, the thermal inactivation kinetics of spores of fourteen strains belonging to the Bacillus subtilis group were determined in detail, using both batch heating in capillary tubes and continuous flow heating in a micro heater. The inactivation data were fitted using a log linear model. Based on the spore heat resistance data, two distinct groups (p subtilis group could be identified. One group of strains had spores with an average D120 °C of 0.33 s, while the spores of the other group displayed significantly higher heat resistances, with an average D120 °C of 45.7 s. When comparing spore inactivation data obtained using batch- and continuous flow heating, the z-values were significantly different, hence extrapolation from one system to the other was not justified. This study clearly shows that heat resistances of spores from different strains in the B. subtilis group can vary greatly. Strains can be separated into two groups, to which different spore heat inactivation kinetics apply. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Comparative characterization of silver nanoparticles synthesized by spore extract of Bacillus subtilis and Geobacillus stearothermophilus

    Directory of Open Access Journals (Sweden)

    Seyed Mahdi Ghasemi

    2018-01-01

    Full Text Available Objective(s: Silver nanostructures have gathered remarkable attention due to their applications in diversefields. Researchers have recently demonstrated that bacterial spores are capable of reducing silver ions toelemental silver leading to formation of nanoparticles.Materials and Methods: In this study, spores of Bacillus subtilis and Geobacillus stearothermophilus wereemployed to produce silver nanoparticles (SNPs from silver nitrate (AgNO3 through a green synthesismethod. The production of SNPs by spores, heat inactivated spores (microcapsule and spore extracts wasmonitored and compared at wavelengths between 300 to 700 nm. The biosynthesized SNPs by spore extractswere characterized and confirmed by XRD and TEM analyses.Results: UV-Visible spectroscopy showed that the spore extracts were able to synthesize more SNPs thanthe other forms. The XRD pattern also revealed that the silver nanometals have crystalline structure withvarious topologies. The TEM micrographs showed polydispersed nanocrystal with dimensions ranging from30 to 90 nm and 15 to 50 nm produced by spore extracts of B. subtilis and G. stearothermophilus, respectively.Moreover, these biologically synthesized nanoparticles exhibited antimicrobial activity against differentopportunistic pathogens.Conclusion: This study suggests the bacterial spore extract as a safe, efficient, cost effective and eco-friendlymaterial for biosynthesis of SNPs.

  20. Bacillus cereus spore damage recovery and diversity in spore germination and carbohydrate utilisation

    NARCIS (Netherlands)

    Warda, Alicja K.

    2016-01-01

    Bacterial spores are extremely robust survival vehicles that are highly resistant towards environmental stress conditions including heat, UV radiation and other stresses commonly applied during food production and preservation. Spores, including those of the toxin-producing food-borne human pathogen

  1. Bacillus cereus spore damage recovery and diversity in spore germination and carbohydrate utilisation

    NARCIS (Netherlands)

    Warda, Alicja K.

    2016-01-01

    Bacterial spores are extremely robust survival vehicles that are highly resistant towards environmental stress conditions including heat, UV radiation and other stresses commonly applied during food production and preservation. Spores, including those of the toxin-producing food-borne human

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

    Science.gov (United States)

    2012-11-01

    Spores of Bacillus species are metabolically dormant and ex-tremely resistant to a wide variety of agents (38). As a conse- quence, these spores can...permeability barrier in dormant spores, the coat is a permeability barrier to large mole- cules (18, 20). Thus, it is possible that there are special...type and gerP spore germina- tion. Almost all bacteria have an alanine racemase activity essen- tial for the generation of the D-alanine needed for

  3. Predictive modeling of Bacillus cereus spores in farm tank milk during grazing and housing periods

    NARCIS (Netherlands)

    Vissers, M.M.M.; Giffel, M.C.T.; Driehuis, F.; Jong, de P.; Lankveld, J.M.G.

    2007-01-01

    The shelf life of pasteurized dairy products depends partly on the concentration of Bacillus cereus spores in raw milk. Based on a translation of contamination pathways into chains of unit-operations, 2 simulation models were developed to quantitatively identify factors that have the greatest effect

  4. Simple, Inexpensive, and Rapid Way to Produce Bacillus subtilis Spores for the Guthrie Bioassay

    Science.gov (United States)

    Franklin, Martha L.; Clark, William A.

    1981-01-01

    Esculin agar has been found to be a simple, inexpensive, rapid, and reliable means to promote production of spores of inhibitor-sensitive clones of Bacillus subtilis strains ATCC 6051 and 6633 for use in the Guthrie bioassay screening tests for genetic metabolic disorders. Images PMID:6790564

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

    NARCIS (Netherlands)

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

    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

  6. 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

  7. Sporulation dynamics and spore heat resistance in wet and dry biofilms of Bacillus cereus

    NARCIS (Netherlands)

    Hayrapetyan, Hasmik; Abee, Tjakko; Nierop Groot, Masja

    2016-01-01

    Environmental conditions and growth history can affect the sporulation process as well as subsequent properties of formed spores. The sporulation dynamics was studied in wet and air-dried biofilms formed on stainless steel (SS) and polystyrene (PS) for Bacillus cereus ATCC 10987 and the

  8. Mutation Induction with UV- and X-radiations in spores and vegetative cells of Bacillus subtilis

    International Nuclear Information System (INIS)

    Tanooka, H.; Munakata, N.; Kitahara, S.

    1978-01-01

    Spores and vegetative cells of Bacillus subtilis strains with various defects in DNA-repair capacities (hcr - , ssp - , hcr - ssp - ) were irradiated with UV radiation or X-rays. Induced mutation frequency was determined from the observed frequency of prototrophic reversion of a suppressible auxotropic mutation. At equal physical dose, after either UV- or X-irradiation, spores were more resistant to mutations as well as to killing than were vegetative cells. However, quantitative comparison revealed that, at equally lethal doses, spores and vegetative cells were almost equally mutable by X-rays whereas spores were considerably less mutable by UV than were vegetative cells. Thus, as judged from their mutagenic efficiency relative to the lethality, X-ray-induced damage in the spore DNA and the vegetative DNA were equally mutagenic, while UV-induced DNA photoproducts in the spore were less mutagenic than those in vegetative cells. Post-treatment of UV-irradiated cells with caffeine decreased the survival and the induced mutation frequency for either spores or vegetative cells for all the strains. In X-irradiated spores however, a similar suppressing effect of caffeine was observed only for mutability of a strain lacking DNA polymerase I activity

  9. Progress in Bacillus subtilis Spore Surface Display Technology towards Environment, Vaccine Development, and Biocatalysis.

    Science.gov (United States)

    Chen, Huayou; Ullah, Jawad; Jia, Jinru

    2017-01-01

    Spore surface display is the most desirable with enhanced effects, low cost, less time consuming and the most promising technology for environmental, medical, and industrial development. Spores have various applications in industry due to their ability to survive in harsh industrial processes including heat resistance, alkaline tolerance, chemical tolerance, easy recovery, and reusability. Yeast and bacteria, including gram-positive and -negative, are the most frequently used organisms for the display of various proteins (eukaryotic and prokaryotic), but unlike spores, they can rupture easily due to nutritive properties, susceptibility to heat, pH, and chemicals. Hence, spores are the best choice to avoid these problems, and they have various applications over nonspore formers due to amenability for laboratory purposes. Various strains of Clostridium and Bacillus are spore formers, but the most suitable choice for display is Bacillus subtilis because, according to the WHO, it is safe to humans and considered as "GRAS" (generally recognized as safe). This review focuses on the application of spore surface display towards industries, vaccine development, the environment, and peptide library construction, with cell surface display for enhanced protein expression and high enzymatic activity. Different vectors, coat proteins, and statistical analyses can be used for linker selection to obtain greater expression and high activity of the displayed protein. © 2017 S. Karger AG, Basel.

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

    Science.gov (United States)

    Warda, Alicja K; den Besten, Heidy M W; Sha, Na; Abee, Tjakko; Nierop Groot, Masja N

    2015-05-18

    Spoilage of heat treated foods can be caused by the presence of surviving spore-formers. It is virtually impossible to prevent contamination at the primary production level as spores are ubiquitous present in the environment and can contaminate raw products. As a result spore inactivation treatments are widely used by food producing industries to reduce the microbial spore loads. However consumers prefer mildly processed products that have less impact on its quality and this trend steers industry towards milder preservation treatments. Such treatments may result in damaged instead of inactivated spores, and these spores may germinate, repair, and grow out, possibly leading to quality and safety issues. The ability to repair and grow out is influenced by the properties of the food matrix. In the current communication we studied the outgrowth from heat damaged Bacillus cereus ATCC 14579 spores on Anopore membrane, which allowed following outgrowth heterogeneity of individual spores on broccoli and rice-based media as well as standard and mildly acidified (pH 5.5) meat-based BHI. Rice, broccoli and BHI pH 5.5 media resulted in delayed outgrowth from untreated spores, and increased heterogeneity compared to BHI pH 7.4, with the most pronounced effect in rice media. Exposure to wet heat for 1 min at 95 °C caused 2 log inactivation and approximately 95% of the spores in the surviving fraction were damaged resulting in substantial delay in outgrowth based on the time required to reach a maximum microcolony size of 256 cells. The delay was most pronounced for heat-treated spores on broccoli medium followed by spores on rice media (both untreated and treated). Interestingly, the increase in outgrowth heterogeneity of heat treated spores on BHI pH 7.4 was more pronounced than on rice, broccoli and BHI pH 5.5 conceivably reflecting that conditions in BHI pH 7.4 better support spore damage repair. This study compares the effects of three main factors, namely heat treatment, p

  11. The immunological characteristics and probiotic function of recombinant Bacillus subtilis spore expressing Clonorchis sinensis cysteine protease.

    Science.gov (United States)

    Tang, Zeli; Shang, Mei; Chen, Tingjin; Ren, Pengli; Sun, Hengchang; Qu, Hongling; Lin, Zhipeng; Zhou, Lina; Yu, Jinyun; Jiang, Hongye; Zhou, Xinyi; Li, Xuerong; Huang, Yan; Xu, Jin; Yu, Xinbing

    2016-12-19

    Clonorchiasis, a food-borne zoonosis, is caused by Clonorchis sinensis. The intestinal tract and bile ducts are crucial places for C. sinensis metacercariae to develop into adult worms. The endospore of Bacillus subtilis is an ideal oral immunization vehicle for delivery of heterologous antigens to intestine. Cysteine protease of C. sinensis (CsCP) is an endogenous key component in the excystment of metacercariae and other physiological or pathological processes. We constructed a fusion gene of CotC (a coat protein)-CsCP and obtained B. subtilis spores with recombinant plasmid of pEB03-CotC-CsCP (B.s-CotC-CsCP). CotC-CsCP expressed on spores' surface was detected by Western blotting and immunofluorescence. Immunological characteristics of recombinant spore coat protein were evaluated in a mouse model. The levels of CsCP-specific antibodies were detected by ELISA. Effects of recombinant spores on mouse intestine were evaluated by histological staining. The activities of biochemical enzymes in serum were assayed by microplate. Liver sections of infected mice were evaluated by Ishak score after Masson's trichrome. The B.s-CotC-CsCP spores displayed CsCP on their coat. Specific IgG and isotypes were significantly induced by coat proteins of B.s-CotC-CsCP spores after subcutaneous immunization. IgA levels in intestinal mucus and bile of B.s-CotC-CsCP orally treated mice significantly increased. Additionally, more IgA-secreting cells were observed in enteraden and lamina propria regions of the mouse jejunum, and an increased amount of acidic mucins in intestines were also observed. There were no significant differences in enzyme levels of serum among groups. No inflammatory injury was observed in the intestinal tissues of each group. The degree of liver fibrosis was significantly reduced after oral immunization with B.s-CotC-CsCP spores. Bacillus subtilis spores maintained the original excellent immunogenicity of CsCP expressed on their surface. Both local and systemic

  12. Effect of Coat Layers in Bacillus Subtilis Spores Resistance to Photo-Catalytic Inactivation

    Directory of Open Access Journals (Sweden)

    Luz del Carmen Huesca-Espitia

    2017-10-01

    Full Text Available Different water treatment processes (physical and chemical exist to obtain safe water for human or food industry supply. The advanced oxidation technologies are rising as a new alternative to eliminate undesirable chemicals and waterborne diseases. In this work, we analyze the power of the photo-assisted Fenton process using Fe(II/H2O2 and UV radiation (365 nm to inactivate Bacillus subtilis spores, considered among the most resistant biological structures known. Different concentrations of Fe(II, H2O2 and UV radiation (365 nm were used to inactivate wt and some coat spore mutants of B. subtilis. Wt spores of B. subtilis were inactivated after 60 min using this process. In general, all defective coat mutants were more sensitive than the wt spores and, particularly, the double mutant was 10 folds more sensitive than others being inactivated during the first 10 minutes using soft reaction conditions. Presence of Fe(II ions was found essential for spore inactivating process and, for those spores inactivated using the Fe(II/H2O2 under UV radiation process, it is suggested that coat structures are important to their resistance to the treatment process. The photo-assisted Fenton process using Fe(II, H2O2 and UV radiation (365 nm can be used to inactivate any water microorganisms with the same or less resistance that B. subtilis spores to produce safe drinking water in relatively short treatment time.

  13. Re-aerosolization of Bacillus thuringiensis spores from concrete and turf.

    Science.gov (United States)

    Bishop, A H; O'Sullivan, C M; Lane, A; Butler Ellis, M C; Sellors, W J

    2017-05-01

    Spores of Bacillus anthracis deposited on surfaces can become airborne again as a result of air currents and mechanical forces. As such, they are a potential source of infection by inhalation. Spores of Bacillus thuringiensis were used to quantify this phenomenon in a simulation of outdoor conditions. Concrete and turf surfaces were inoculated by aerosol to produce high spore densities (greater than 1 × 10 9  CFU per m 2 ) which were then subjected to the passage of air at 10 ms -1 with and without simulated walking. Re-aerosolized spores were sampled by wetted wall cyclone air samplers. The mean total re-aerosolization rate from concrete (m -2  min -1 ) was 1·16 × 10 -3 for wind alone and 3·2 × 10 -3 for wind and simulated walking while for turf the respective values were 2·7 × 10 -4 and 6·7 × 10 -4 . Following the malicious and/or accidental release of an aerosol of Bacillus anthracis spores, the immediate risk of human inhalation would decrease as the spores were deposited on surfaces or diluted by wind flow. There is, however, a concern that the deposited spores could become re-aerosolized and so present an ongoing hazard. Using an accurate simulant for B. anthracis spores a method is reported here that allowed the enumeration of re-aerosolized spores from concrete and turf by wind flow and footfall. Under the conditions used, the rates of re-aerosolization were low. These findings will need to be verified under real outdoor conditions before the true significance in terms of secondary exposure to pathogenic spores can be assessed. © 2017 Crown copyright. Letter of Applied Microbiology © 2017 The Society for Applied Microbiology. This article is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland.

  14. 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 (T 0 ) 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.

  15. A four-gene operon inBacillus cereusproduces two rare spore-decorating sugars.

    Science.gov (United States)

    Li, Zi; Mukherjee, Thiya; Bowler, Kyle; Namdari, Sholeh; Snow, Zachary; Prestridge, Sarah; Carlton, Alexandra; Bar-Peled, Maor

    2017-05-05

    Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3- C -methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM: C -methyltransferase, and NADPH-dependent CDP-3- C -methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3- C -methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3- C -methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3- C -methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C -methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2-1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3- C -methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. 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

    2014-01-01

    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

  17. BACILLUS ATROPHAEUS SKD – 1 КАК ПЕРСПЕКТИВНЫЙ ШТАММ ДЛЯ РАЗРАБОТКИ БИОПРЕПАРАТА

    Directory of Open Access Journals (Sweden)

    M. F. Koryazhkina

    2010-01-01

    Full Text Available Strain Bacillus atrophaeus is allocated was isolated from soils of Astrakhan region. Strain has complex of economicuseful properties and is promising to create a biological product for protection of plants against diseases and stimulation of growth. Antifungal and phytostimulating properties of the strain were studied in laboratory experiments. Researches the ability of strain to the stimulation of plants growth were conducted in the vegetative experiment on tomatoes.

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

    Science.gov (United States)

    2014-01-01

    Microbiol 102, 65 76. Butzin, X.Y., Troiano, A.J., Coleman , W.H., Griffiths , K.K., Doona, C.J., Feeherry, F.E., Wang, G., Li, Y. Q. et al. (2012...germination, possibly because it is essential for organization of GRs in a complex in spores’ inner membrane ( Griffiths et aL 2011). Journal of... Griffiths , K.K., Zhang, J., Cowan, A.E., Yu, J. and Setlow, P. (2011) Germination proteins in the inner membrane of dormant Bacillus subtilis spores

  19. Small, acid-soluble proteins bound to DNA protect Bacillus subtilis spores from killing by dry heat.

    OpenAIRE

    Setlow, B; Setlow, P

    1995-01-01

    Dry Bacillus subtilis spores lacking their two major DNA-binding proteins (small, acid-soluble proteins [SASP] alpha and beta) were much more sensitive to dry heat than were wild-type spores. Survivors of dry heat treatment of both wild-type and mutant spores exhibited a high frequency of mutations, and the DNA from the heated spores had increased numbers of single-strand breaks. These data indicate that SASP alpha and beta provide significant protection to spore DNA against the damaging effe...

  20. Laboratory Investigations on the Survival of Bacillus subtilis Spores in Deliquescent Salt Mars Analog Environments

    Science.gov (United States)

    Nuding, Danielle L.; Gough, Raina V.; Venkateswaran, Kasthuri J.; Spry, James A.; Tolbert, Margaret A.

    2017-10-01

    Observed features such as recurring slope lineae suggest that liquid water may exist on the surface and near-subsurface of Mars today. The presence of this liquid water, likely in the form of a brine, has important implications for the present-day water cycle, habitability, and planetary protection policies. It is possible that this water is formed, at least partially, by deliquescence of salts, a process during which hygroscopic salts absorb water vapor from the atmosphere and form a saturated liquid brine. We performed laboratory experiments to examine the ability of Bacillus subtilis (B-168) spores, alone or mixed with calcium perchlorate salt (Ca(ClO4)2), to form liquid water via deliquescence under Mars-relevant conditions. Spore survival after exposure to these conditions was examined. An environmental chamber was used to expose the samples to temperature and relative humidity (RH) values similar to those found on Mars, and Raman microscopy was used to identify the phases of water and salt that were present and to confirm the presence of spores. We found that B-168 spores did not condense any detectable water vapor on their own during the diurnal cycle, even at 100% RH. However, when spores were mixed with perchlorate salt, the entire sample deliquesced at low RH values, immersing the spores in a brine solution during the majority of the simulated martian temperature and humidity cycle. After exposure to the simulated diurnal cycles and, in some cases, perchlorate brine, the impact of each environmental scenario on spore survival was estimated by standard plate assay. We found that, if there are deliquescent salts in contact with spores, there is a mechanism for the spores to acquire liquid water starting with only atmospheric water vapor as the H2O source. Also, neither crystalline nor liquid Ca(ClO4)2 is sporicidal despite the low water activity.

  1. 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.

  2. Sensitivity of Spores of Eight Bacillus Cereus Strains to Pressure-Induced Germination by Moderate Hydrostatic Pressure, Time and Temperature

    National Research Council Canada - National Science Library

    Kalchayanand, Norasak; Ray, Bibek; Dunne, C. P; Sikes, Anthony

    2005-01-01

    The spores of eight Bacillus cereus strains were pressurized at 138 to 483 MPa for 5 to 20 min at 25 to 70 C in order to determine the sensitive and the resistant strains to pressure-induced germination...

  3. Heat resistance of Bacillus spores : Natural variation and genomic adaptation

    NARCIS (Netherlands)

    Berendsen, Erwin Mathijs

    2016-01-01

    Ons onderzoek heeft zich gericht op de hitteresistentie van sporen van verschillende Bacillus soorten. Het is een bekend probleem dat sporen van bepaalde stammen hittebehandelingen die worden toegepast bij voedselproductie kunnen overleven. In ons onderzoek hebben we allereerst aangetoond dat er

  4. Detection of Bacillus anthracis spores from environmental water using bioluminescent reporter phage.

    Science.gov (United States)

    Nguyen, C; Makkar, R; Sharp, N J; Page, M A; Molineux, I J; Schofield, D A

    2017-11-01

    We investigated the ability of a temperate Bacillus anthracis reporter phage (Wβ::luxAB-2), which transduces bioluminescence to infected cells, to detect viable spores from deliberately contaminated environmental water samples. Environmental water was inoculated with spores and assayed with Wβ::luxAB-2. Bioluminescent signals directly correlated with input phage and spore concentrations. A limit of detection of 10 1 and 10 2 CFU per ml within 8 h was achieved from pond and lake water, respectively. Detection was greatly simplified by minimizing sample processing steps without spore extraction. The complex endogenous microbial flora and salt content of brackish water challenged the assay, extending the detection time to 12 h for a sensitivity of 10 2 CFU per ml. Phage-mediated bioluminescence was strictly dependent on bacterial physiology, being significantly reduced in mid/late log phase cells. This was shown to be due to an inability of the phage to adsorb. The reporter phage Wβ::luxAB-2 displays potential for simplified detection of viable spores from contaminated water samples within 12 h. A deliberate aerosol release of spores could lead to widespread contamination, leaving large areas uninhabitable until remediation. An essential requirement of this restoration process is the development of simplified detection assays in different environmental matrices. © 2017 The Society for Applied Microbiology.

  5. Anthrax surrogate spores are destroyed by PDT mediated by phenothiazinium dyes

    Science.gov (United States)

    Demidova, Tatiana N.; Hamblin, Michael R.

    2005-04-01

    Some Gram-positive bacteria (including the causative agent of anthrax - Bacillus anthracis) survive conditions of stress and starvation by producing dormant stage spores. The spore"s multilayered capsule consists of inner and outer membranes, cortex, proteinaceous spore coat, and in some species an exosporium. These outer layers enclose dehydrated and condensed DNA, saturated with small, acid-soluble proteins. These protective structures make spores highly resistant to damage by heat, radiation, and commonly employed anti-bacterial agents. Previously Bacillus spores have been shown to be resistant to photodynamic inactivation (PDI) using dyes and light that easily destroy the corresponding vegetative bacteria, but recently we have discovered that they are susceptible to PDI. Photoinactivation, however, is only possible if phenothiazinium dyes are used. Dimethylmethylene blue, methylene blue, new methylene blue and toluidine blue O are all effective photosensitizers. Alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin and benzoporphyrin derivative are ineffective against spores even though they can easily kill vegetative cells. Spores of B. cereus and B. thuringiensis are most susceptible, B. subtilis and B. atrophaeus are also killed, while B. megaterium is resistant. Photoinactivation is most effective when excess dye is washed from the spores showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores and for which conventional sporicides would have unacceptable tissue toxicity.

  6. 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

  7. Novel Sample Preparation Method for Safe and Rapid Detection of Bacillus anthracis Spores in Environmental Powders and Nasal Swabs

    OpenAIRE

    Luna, Vicki A.; King, Debra; Davis, Carisa; Rycerz, Tony; Ewert, Matthew; Cannons, Andrew; Amuso, Philip; Cattani, Jacqueline

    2003-01-01

    Bacillus anthracis spores have been used as a biological weapon in the United States. We wanted to develop a safe, rapid method of sample preparation that provided safe DNA for the detection of spores in environmental and clinical specimens. Our method reproducibly detects B. anthracis in samples containing

  8. Characterization of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores: Phenotype and transcriptome analysis

    NARCIS (Netherlands)

    Melis, van C.C.J.; Nierop Groot, M.N.; Tempelaars, M.H.; Moezelaar, R.; Abee, T.

    2011-01-01

    Sorbic acid (SA) is widely used as a preservative, but the effect of SA on spore germination and outgrowth has gained limited attention up to now. Therefore, the effect of sorbic acid on germination of spores of Bacillus cereus strain ATCC 14579 was analyzed both at phenotype and transcriptome

  9. 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.

  10. Live cell imaging of germination and outgrowth of individual bacillus subtilis spores; the effect of heat stress quantitatively analyzed with SporeTracker.

    Directory of Open Access Journals (Sweden)

    Rachna Pandey

    Full Text Available Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program "SporeTracker" allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less and fewer grew out (48.4% less after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased and the distribution and average of the duration of germination itself (increased. However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.

  11. Inactivation of Bacillus subtilis spores by combined pulsed light and thermal treatments.

    Science.gov (United States)

    Artíguez, Mari Luz; Martínez de Marañón, Iñigo

    2015-12-02

    The combined effect of pulsed light (PL) and heat processing was evaluated on the inactivation of Bacillus subtilis spores. Those processes were applied separately and the time between both treatments was modified to evaluate whether the effect of the first treatment is maintained for a long time. B. subtilis spores subjected to sublethal pre-treatments were more sensitive to subsequent treatments (PL or thermal treatments) than untreated spores. Heating followed by PL was the most effective combination in reducing B. subtilis counts. Bacterial spores remained sensitized to subsequent treatment for at least 24 h of storage in water, whatever the temperature was (4 or 30°C). Sensitivity of B. subtilis cells to PL or heat processing increased after germination in a nutrient broth, being equally sensitive from 3 to 24 h. Vegetative cells maintained their enhanced sensitivity to subsequent processing after spore germination. The results of this work demonstrate that the combination of heating and PL treatment is a promising preservation method for microbial inactivation. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Development of bioprocess for high density cultivation yield of the probiotic Bacillus coagulans and its spores

    Directory of Open Access Journals (Sweden)

    Kavita R. Pandey

    2016-09-01

    Full Text Available Bacillus coagulans is a spore forming lactic acid bacterium. Spore forming bacteria, have been extensively studied and commercialized as probiotics. Probiotics are produced by fermentation technology. There is a limitation to biomass produced by conventional modes of fermentation. With the great demand generated by range of probiotic products, biomass is becoming very valuable for several pharmaceutical, dairy and probiotic companies. Thus, there is a need to develop high cell density cultivation processes for enhanced biomass accumulation. The bioprocess development was carried out in 6.6 L bench top lab scale fermentor. Four different cultivation strategies were employed to develop a bioprocess for higher growth and sporulation efficiencies of probiotic B. coagulans. Batch fermentation of B. coagulans yielded 18 g L-1 biomass (as against 8.0 g L-1 productivity in shake flask with 60% spore efficiency. Fed-batch cultivation was carried out for glucose, which yielded 25 g L-1 of biomass. C/N ratio was very crucial in achieving higher spore titres. Maximum biomass yield recorded was 30 g L-1, corresponding to 3.8 × 1011 cells mL-1 with 81% of cells in sporulated stage. The yield represents increment of 85 times the productivity and 158 times the spore titres relative to the highest reported values for high density cultivation of B. coagulans.

  13. Clostridium thermocellum Nitrilase Expression and Surface Display on Bacillus subtilis Spores.

    Science.gov (United States)

    Chen, Huayou; Zhang, Tianxi; Sun, Tengyun; Ni, Zhong; Le, Yilin; Tian, Rui; Chen, Zhi; Zhang, Chunxia

    2015-01-01

    Nitrilases are an important class of industrial enzymes. They require mild reaction conditions and are highly efficient and environmentally friendly, so they are used to catalyze the synthesis of carboxylic acid from nitrile, a process considered superior to conventional chemical syntheses. Nitrilases should be immobilized to overcome difficulties in recovery after the reaction and to stabilize the free enzyme. The nitrilase from Clostridium thermocellum was expressed, identified and displayed on the surface of Bacillus subtilis spores by using the spore coat protein G of B. subtilis as an anchoring motif. In a free state, the recombinant nitrilase catalyzed the conversion of 3-cyanopyridine to niacin and displayed maximum catalytic activity (8.22 units/mg protein) at 40 °C and pH 7.4. SDS-PAGE and Western blot were used to confirm nitrilase display. Compared with the free enzyme, the spore-immobilized nitrilase showed a higher tolerance for adverse environmental conditions. After the reaction, recombinant spores were recovered via centrifugation and reused 3 times to catalyze the conversion of 3-cyanopyridine with 75.3% nitrilase activity. This study demonstrates an effective means of nitrilase immobilization via spore surface display, which can be applied in biological processes or conversion. © 2015 S. Karger AG, Basel.

  14. 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.

  15. 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.

  16. 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.

    2016-01-01

    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, 102, 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. PMID:27213119

  17. Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Tomasz Łęga

    Full Text Available Effective vaccination against influenza virus infection is a serious problem mainly due to antigenic variability of the virus. Among many of investigated antigens, the extracellular domain of the M2 protein (M2e features high homology in all strains of influenza A viruses and antibodies against M2e and is protective in animal models; this makes it a potential candidate for generation of a universal influenza vaccine. However, due to the low immunogenicity of the M2e, formulation of a vaccine based on this antigen requires some modification to induce effective immune responses. In this work we evaluated the possible use of Bacillus subtilis spores as a carrier of the Influenza A M2e antigen in mucosal vaccination. A tandem repeat of 4 consensus sequences coding for human-avian-swine-human M2e (M2eH-A-S-H peptide was fused to spore coat proteins and stably exposed on the spore surface, as demonstrated by the immunostaining of intact, recombinant spores. Oral immunization of mice with recombinant endospores carrying M2eH-A-S-H elicited specific antibody production without the addition of adjuvants. Bacillus subtilis endospores can serve as influenza antigen carriers. Recombinant spores constructed in this work showed low immunogenicity although were able to induce antibody production. The System of influenza antigen administration presented in this work is attractive mainly due to the omitting time-consuming and cost-intensive immunogen production and purification. Therefore modification should be made to increase the immunogenicity of the presented system.

  18. 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

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

    Science.gov (United States)

    2008-03-01

    spores were grown in plastic petri dishes on Criterion Dehydrated Culture Media , which contained per liter of formula 15 grams agar , 5 grams gelatin...became reality in 2001 when terrorists sent spores in a powdered form in letters to two senators and several news media offices, killing five people...is the causative agent of the disease anthrax. B. thuringiensis is often used in pesticides and bioengineering pest resistant crops because of its

  20. 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.

  1. Response surface modeling for hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores

    Science.gov (United States)

    2014-01-01

    Response surface methodology using a face-centered cube design was used to describe and predict spore inactivation of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure of six spore-contaminated materials to hot, humid air. For each strain/material pair, an attempt was made to fit a first or second order model. All three independent predictor variables (temperature, relative humidity, and time) were significant in the models except that time was not significant for B. thuringiensis Al Hakam on nylon. Modeling was unsuccessful for wiring insulation and wet spores because there was complete spore inactivation in the majority of the experimental space. In cases where a predictive equation could be fit, response surface plots with time set to four days were generated. The survival of highly purified Bacillus spores can be predicted for most materials tested when given the settings for temperature, relative humidity, and time. These predictions were cross-checked with spore inactivation measurements. PMID:24949256

  2. 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.

  3. Changes in ultraviolet resistance and photoproduct formation as early events in spore germination of Bacillus cereus T

    International Nuclear Information System (INIS)

    Irie, R.

    1978-01-01

    In order to determine the timing of the change in the state of DNA in bacterial spores during the course of germination, L-alanine-induced germination of Bacillus cereus spores was interrupted by 0.3M CaCl 2 as an inhibitor, and the resulting semi-refractive spores (spores at the end of the first phase of germination) were examined for UV-resistance and photoproduct formation. Upon UV-irradiation, these spores, still having a semi-refractile core as observed under a phase-contrast microscope, gave rise to mainly the cyclobutane-type thymine dimer. It was concluded that change in the stats of the spore DNA occurs early in the process of germination, i.e. before the refractility of the core is lost. It was also found that CaCl 2 markedly prolonged the duration of the transient UV-resistant stage. (author)

  4. Low cost medium for spore production of Bacillus KKU02 and KKU03 and the effects of the produced spores on growth of giant freshwater prawn (Macrobrachium rosenbergii de Man).

    Science.gov (United States)

    Wangka-Orm, Charkrit; Deeseenthum, Sirirat; Leelavatcharamas, Vichai

    2014-08-01

    In order to extend the shelf life of 2 high potential Bacillus probiotic isolates which were Bacillus KKU02 and Bacillus KKU03, the spore forms of these 2 Bacillus isolates were studied for using as probiotic instead. The low cost medium for spore production of these 2 Bacillus isolates was examined in order to produce probiotic spores for feeding the shrimps. It was found that cassava at 100 g L(-1) and supplemented with 20.0 g L(-1) dextrose, 0.1 g L(-1) MgSO4 and 2.0 g L(-1) (NH4)2SO4 showed the highest spore concentration at about 1 x 10(8) CFU mL(-1). The effects of feeding these 2 Bacillus spores on the growth of giant freshwater prawns were further examined. The spores of Bacillus KKU02 and Bacillus KKU03 (-10(7) spore mL(-1)) in pure and mixed culture forms were mixed with commercial prawn feed (200 mL kg(-1)) to give six feed treatments. Body length and weight of the prawns in mixed spore culture tanks after rearing for 90 days (13.5 cm and 59.8 g, respectively) were significantly higher (p = 0.05) than other treatments. The treated prawns were further challenged with Aeromonas hydrophila for 7 days. The percentages of survival after the challenge in the prawns fed with the mixed spores (46.8%) were also found significantly higher (p = 0.05) than others groups, except the mixed live cell treatment (60%). These results indicated that the spores of Bacillus KKU02 and Bacillus KKU03 had a high potential for using as commercial probiotics.

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

    Science.gov (United States)

    From, Cecilie; van der Voort, Menno; Abee, Tjakko; Granum, Per Einar

    2012-06-01

    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 of the gene in this group of bacteria. Quantitative PCR revealed that bc1245 is transcribed late in sporulation (upon formation of phase-bright spores) and at the same time as the mother cell-specific transcription factor σ(K) . The σ(K) regulon includes structural components of the spore (such as coat proteins), and it is therefore plausible that bc1245 might encode a structural outer spore protein. This was confirmed by detection of BC1245 in exosporium extracts from B. cereus by immunoblotting against BC1245 antiserum. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  6. Mucosal vaccine delivery by non-recombinant spores of Bacillus subtilis.

    Science.gov (United States)

    Ricca, Ezio; Baccigalupi, Loredana; Cangiano, Giuseppina; De Felice, Maurilio; Isticato, Rachele

    2014-08-12

    Development of mucosal vaccines strongly relies on an efficient delivery system and, over the years, a variety of approaches based on phages, bacteria or synthetic nanoparticles have been proposed to display and deliver antigens. The spore of Bacillus subtilis displaying heterologous antigens has also been considered as a mucosal vaccine vehicle, and shown able to conjugate some advantages of live microrganisms with some of synthetic nanoparticles. Here we review the use of non-recombinant spores of B. subtilis as a delivery system for mucosal immunizations. The non-recombinant display is based on the adsorption of heterologous molecules on the spore surface without the need of genetic manipulations, thus avoiding all concerns about the use and environmental release of genetically modified microorganisms. In addition, adsorbed molecules are stabilized and protected by the interaction with the spore, suggesting that this system could reduce the rapid degradation of the antigen, often observed with other delivery systems and identified as a major drawback of mucosal vaccines.

  7. The effect of recovery conditions on the apparent heat resistance of Bacillus cereus spores.

    Science.gov (United States)

    Gonzalez, I; Lopez, M; Mazas, M; Gonzalez, J; Bernardo, A

    1995-05-01

    The effect of recovery media and incubation temperature on the apparent heat resistance of three ATCC strains (4342, 7004 and 9818) of Bacillus cereus spores were studied. Nutrient Agar (NA), Tryptic Soy Agar (TSA), Plate Count Agar (PCA) and Milk Agar (MA) as the media and temperatures in the range of 15-40 degrees C were used to recover heated spores. Higher counts of heat injured spores were obtained on PCA and NA. The optimum subculture temperature was about 5 degrees C below the optimum temperature for unheated spores. No significant differences in heat resistance were observed with the different recovery conditions except for strains 4342 and 9818 when MA was used as plating medium. Large differences in D-values were found among the strains (D100 = 0.28 min for 7004; D100 = 0.99 min for 4342; D100 = 4.57 min for 9818). The 7004 strain showed a sub-population with a greater heat resistance. The z values obtained for the three strains studied under the different recovery conditions were similar (7.64 degrees C +/- 0.25).

  8. 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.

  9. High-Level Heat Resistance of Spores of Bacillus amyloliquefaciens and Bacillus licheniformis Results from the Presence of a spoVA Operon in a Tn1546 Transposon

    Science.gov (United States)

    Berendsen, Erwin M.; Koning, Rosella A.; Boekhorst, Jos; de Jong, Anne; Kuipers, Oscar P.; Wells-Bennik, Marjon H. J.

    2016-01-01

    Bacterial endospore formers can produce spores that are resistant to many food processing conditions, including heat. Some spores may survive heating processes aimed at production of commercially sterile foods. Recently, it was shown that a spoVA operon, designated spoVA2mob, present on a Tn1546 transposon in Bacillus subtilis, leads to profoundly increased wet heat resistance of B. subtilis spores. Such Tn1546 transposon elements including the spoVA2mob operon were also found in several strains of Bacillus amyloliquefaciens and Bacillus licheniformis, and these strains were shown to produce spores with significantly higher resistances to wet heat than their counterparts lacking this transposon. In this study, the locations and compositions of Tn1546 transposons encompassing the spoVA2mob operons in B. amyloliquefaciens and B. licheniformis were analyzed. Introduction of these spoVA2mob operons into B. subtilis 168 (producing spores that are not highly heat resistant) rendered mutant 168 strains that produced high-level heat resistant spores, demonstrating that these elements in B. amyloliquefaciens and B. licheniformis are responsible for high level heat resistance of spores. Assessment of growth of the nine strains of each species between 5.2°C and 57.7°C showed some differences between strains, especially at lower temperatures, but all strains were able to grow at 57.7°C. Strains of B. amyloliquefaciens and B. licheniformis that contain the Tn1546 elements (and produce high-level heat resistant spores) grew at temperatures similar to those of their Tn1546-negative counterparts that produce low-level heat resistant spores. The findings presented in this study allow for detection of B. amyloliquefaciens and B. licheniformis strains that produce highly heat resistant spores in the food chain. PMID:27994575

  10. High-level heat resistance of spores of Bacillus amyloliquefaciens and Bacillus licheniformis results from the presence of a spoVA operon in a Tn1546 transposon.

    Directory of Open Access Journals (Sweden)

    Erwin M. Berendsen

    2016-12-01

    Full Text Available Bacterial endospore formers can produce spores that are resistant to many food processing conditions, including heat. Some spores may survive heating processes aimed at production of commercially sterile foods. Recently, it was shown that a spoVA operon, designated spoVA2mob, present on a Tn1546 transposon in Bacillus subtilis, leads to profoundly increased wet heat resistance of B. subtilis spores. Such Tn1546 transposon elements including the spoVA2mob operon were also found in several strains of Bacillus amyloliquefaciens and Bacillus licheniformis, and these strains were shown to produce spores with significantly higher resistances to wet heat than their counterparts lacking this transposon. In this study, the locations and compositions of Tn1546 transposons encompassing the spoVA2mob operons in B. amyloliquefaciens and B. licheniformis were analyzed. Introduction of these spoVA2mob operons into B. subtilis 168 (producing spores that are not highly heat resistant rendered mutant 168 strains that produced high-level heat resistant spores, demonstrating that these elements in B. amyloliquefaciens and B. licheniformis are responsible for high level heat resistance of spores. Assessment of growth of the nine strains of each species between 5.2°C and 57.7°C showed some differences between strains, especially at lower temperatures, but all strains were able to grow at 57.7°C. Strains of B. amyloliquefaciens and B. licheniformis that contain the Tn1546 elements (and produce high-level heat resistant spores grew at temperatures similar to those of their Tn1546-negative counterparts that produce low-level heat resistant spores. The findings presented in this study allow for detection of B. amyloliquefaciens and B. licheniformis strains that produce highly heat resistant spores in the food chain.

  11. 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.

  12. Effects of solar ultraviolet radiations on Bacillus subtilis spores and T-7 bacteriophage

    Science.gov (United States)

    Spizizen, J.; Isherwood, J. E.; Taylor, G. R.

    1975-01-01

    Spores of Bacillus subtilis HA 101 and the DNA polymerase I-defective mutant HA 101 (59)F were exposed to selected wavelengths of solar ultraviolet light and space vacuum during the return of Apollo 16. In addition, coliphage T-7 suspensions were exposed to solar ultraviolet radiation as part of the Microbial Response to Space Environment Experiment. Optical filters were employed to provide different energy levels at wavelengths 254 nm and 280 nm. Dose-response curves for lethal and mutagenic effects were compared with ground-based data. A close parallel was observed between the results of solar radiation and ground tests with spores of the two strains. However, significantly greater inactivation of T-7 bacteriophage was observed after exposure to solar ultraviolet radiation.

  13. Thermal resistance of Bacillus stearothermophilus spores in different heating systems containing some approved food additives.

    Science.gov (United States)

    López, M; Mazas, M; González, I; González, J; Bernardo, A

    1996-09-01

    The effects of different heating systems on the heat resistance of Bacillus stearothermophilus spores (ATCC 7953, 12980, 15951 and 15952) were investigated. Spores were heated in distilled water, Sorensen buffer (0.18 mol 1-1), McIlvaine buffer (0.0025-0.18 mol 1-1), and several solutions containing sodium chloride (0.06-12%), sodium nitrite (125 ppm), potassium sorbate (0.1%) and sodium benzoate (0.1%) over a wide range of temperatures (115-140 degrees C). D-values obtained for McIlvaine and Sorensen buffers, at the same molarities, were not significantly different (P > 0.05), but decimal reduction times increased as phosphate concentrations in the solutions decreased. The concentrations, in which statistically significant differences (P 0.05).

  14. 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.

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

    OpenAIRE

    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 microorganisms such as Bacillus cereus, that may survive the use of milder heating regimes. Dormant spores produced by B. cereus can survive processing conditions and their subsequent outgrowth increases ...

  16. Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples.

    Science.gov (United States)

    Thomas, M C; Shields, M J; Hahn, K R; Janzen, T W; Goji, N; Amoako, K K

    2013-07-01

    Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10-fold serial dilutions of Bacillus anthracis spores using quantitative real-time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10(1) and 1·3 × 10(2)  CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS). The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors. Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit. The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples. © Her Majesty the Queen in Right of Canada [2013]. Reproduced with the permission of the Canadian Food Inspection Agency.

  17. The effect of metal ions commonly present in food on gene expression of sporulating Bacillus subtilis cells in relation to spore wet heat resistance.

    NARCIS (Netherlands)

    Oomes, S.J.C.M.; Brul, S.

    2004-01-01

    Bacillus subtilis is a food spoilage spore-forming bacterium. The spores can be very heat-resistant and may cause problems in the production of foods. Varying the metal concentration in the sporulation media is known to influence the heat resistance of the spores. The effect of changing the metal

  18. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Role of the gerA operon in L-alanine germination of Bacillus licheniformis spores

    Directory of Open Access Journals (Sweden)

    Løvdal Irene S

    2012-03-01

    Full Text Available Abstract Background The genome of Bacillus licheniformis DSM 13 harbours three neighbouring open reading frames showing protein sequence similarities to the proteins encoded from the Bacillus subtilis subsp. subtilis 168 gerA operon, GerAA, GerAB and GerAC. In B. subtilis, these proteins are assumed to form a germinant receptor involved in spore germination induced by the amino acid L-alanine. Results In this study we show that disruption of the gerAA gene in B. licheniformis MW3 hamper L-alanine and casein hydrolysate-triggered spore germination, measured by absorbance at 600 nm and confirmed by phase contrast microscopy. This ability was restored by complementation with a plasmid-borne copy of the gerA locus. Addition of D-alanine in the casein hydrolysate germination assay abolished germination of both B. licheniformis MW3 and the complementation mutant. Germination of both B. licheniformis MW3 and the gerA disruption mutant was induced by the non-nutrient germinant Ca2+-Dipicolinic acid. Conclusions These results demonstrate that the B. licheniformis MW3 gerA locus is involved in germination induced by L-alanine and potentially other components present in casein hydrolysate.

  20. Role of the gerA operon in L-alanine germination of Bacillus licheniformis spores

    Science.gov (United States)

    2012-01-01

    Background The genome of Bacillus licheniformis DSM 13 harbours three neighbouring open reading frames showing protein sequence similarities to the proteins encoded from the Bacillus subtilis subsp. subtilis 168 gerA operon, GerAA, GerAB and GerAC. In B. subtilis, these proteins are assumed to form a germinant receptor involved in spore germination induced by the amino acid L-alanine. Results In this study we show that disruption of the gerAA gene in B. licheniformis MW3 hamper L-alanine and casein hydrolysate-triggered spore germination, measured by absorbance at 600 nm and confirmed by phase contrast microscopy. This ability was restored by complementation with a plasmid-borne copy of the gerA locus. Addition of D-alanine in the casein hydrolysate germination assay abolished germination of both B. licheniformis MW3 and the complementation mutant. Germination of both B. licheniformis MW3 and the gerA disruption mutant was induced by the non-nutrient germinant Ca2+-Dipicolinic acid. Conclusions These results demonstrate that the B. licheniformis MW3 gerA locus is involved in germination induced by L-alanine and potentially other components present in casein hydrolysate. PMID:22420404

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

    Energy Technology Data Exchange (ETDEWEB)

    Kweon, Hyojin [Georgia Inst. of Technology, Atlanta, GA (United States); Yiacoumi, Sotira [Georgia Inst. of Technology, Atlanta, GA (United States); Tsouris, Costas [Georgia Inst. of Technology, Atlanta, GA (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

    2011-11-07

    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. In conclusion, 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.

  2. Predicting Bacillus coagulans spores inactivation in tomato pulp under nonisothermal heat treatments.

    Science.gov (United States)

    Zimmermann, Morgana; Longhi, Daniel A; Schaffner, Donald W; Aragão, Gláucia M F

    2014-05-01

    The knowledge and understanding of Bacillus coagulans inactivation during a thermal treatment in tomato pulp, as well as the influence of temperature variation during thermal processes are essential for design, calculation, and optimization of the process. The aims of this work were to predict B. coagulans spores inactivation in tomato pulp under varying time-temperature profiles with Gompertz-inspired inactivation model and to validate the model's predictions by comparing the predicted values with experimental data. B. coagulans spores in pH 4.3 tomato pulp at 4 °Brix were sealed in capillary glass tubes and heated in thermostatically controlled circulating oil baths. Seven different nonisothermal profiles in the range from 95 to 105 °C were studied. Predicted inactivation kinetics showed similar behavior to experimentally observed inactivation curves when the samples were exposed to temperatures in the upper range of this study (99 to 105 °C). Profiles that resulted in less accurate predictions were those where the range of temperatures analyzed were comparatively lower (inactivation profiles starting at 95 °C). The link between fail prediction and both lower starting temperature and magnitude of the temperature shift suggests some chemical or biological mechanism at work. Statistical analysis showed that overall model predictions were acceptable, with bias factors from 0.781 to 1.012, and accuracy factors from 1.049 to 1.351, and confirm that the models used were adequate to estimate B. coagulans spores inactivation under fluctuating temperature conditions in the range from 95 to 105 °C. How can we estimate Bacillus coagulans inactivation during sudden temperature shifts in heat processing? This article provides a validated model that can be used to predict B. coagulans under changing temperature conditions. B. coagulans is a spore-forming bacillus that spoils acidified food products. The mathematical model developed here can be used to predict the spoilage

  3. Surface display of bacterial tyrosinase on spores of Bacillus subtilis using CotE as an anchor protein.

    Science.gov (United States)

    Hosseini-Abari, Afrouzossadat; Kim, Byung-Gee; Lee, Sang-Hyuk; Emtiazi, Giti; Kim, Wooil; Kim, June-Hyung

    2016-12-01

    Tyrosinases, copper-containing monooxygenases, are widely used enzymes for industrial, medical, and environmental applications. We report the first functional surface display of Bacillus megaterium tyrosinase on Bacillus subtilis spores using CotE as an anchor protein. Flow Cytometry was used to verify surface expression of tyrosinase on the purified spores. Moreover, tyrosinase activity of the displayed enzyme on B. subtilis spores was monitored in the presence of L-tyrosine (substrate) and CuSO 4 (inducer). The stability of the spore-displayed tyrosinase was then evaluated after 15 days maintenance of the spores at room temperature, and no significant decrease in the enzyme activity was observed. In addition, the tyrosinase-expressing spores could be repeatedly used with 62% retained enzymatic activity after six times washing with Tris-HCl buffer. This genetically immobilized tyrosinase on the spores would make a new advance in industrial, medical, and environmental applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. 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.

  5. 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.

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

    Science.gov (United States)

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

    2010-07-01

    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. Tempe extract showed a high antibacterial activity against B. cereus ATCC 14579 based on optical density and viable count measurements. This growth inhibition was manifested by a 4 log CFU ml(-1) reduction, within the first 15 min of exposure. Tempe extracts also rapidly inactivated B. cereus spores upon germination. Viability and membrane permeability assessments using fluorescence probes showed rapid inactivation and permeabilization of the cytoplasmic membrane confirming the bactericidal mode of action. Cooked beans and Rhizopus grown on different media did not show antibacterial activity, indicating the unique association of the antibacterial activity with tempe. Subsequent characterization of the antibacterial activity revealed that heat treatment and protease addition nullified the bactericidal effect, indicating the proteinaceous nature of the bioactive compound. During fermentation of soya beans with Rhizopus, compounds are released with extensive antibacterial activity against B. cereus cells and spores. The results show the potential of producing natural antibacterial compounds that could be used as ingredients in food preservation and pathogen control. © 2009 The Authors. Journal compilation © 2009 The Society for Applied Microbiology.

  7. 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.

  8. Low persistence of Bacillus thuringiensis serovar israelensis spores in four mosquito biotopes of a salt marsh in southern France.

    Science.gov (United States)

    Hajaij, Myriam; Carron, Alexandre; Deleuze, Julien; Gaven, Bruno; Setier-Rio, Marie-Laure; Vigo, Gerard; Thiéry, Isabelle; Nielsen-LeRoux, Christina; Lagneau, Christophe

    2005-11-01

    We studied the persistence of Bacillus thuringiensis serovar israelensis (Bti) in a typical breeding site of the mosquito Ochlerotatus caspius in a particularly sensitive salt marsh ecosystem following two Bti-based larvicidal applications (Vectobac 12AS, 1.95 L/ha). The treated area was composed of four larval biotopes that differed in terms of the most representative plant species (Sarcocornia fruticosa, Bolboschoenus maritimus, Phragmites australis, and Juncus maritimus) and the physical and chemical characteristics of the soil. We sampled water, soil, and plants at various times before and after the applications (from spring to autumn, 2001) and quantified the spores of B. thuringiensis (Bt) and Bacillus species. The B. cereus group accounted for between 0% and 20% of all Bacillus spp. before application depending on the larval biotope. No Bti were found before application. The variation in the quantity of bacilli during the mosquito breeding season depended more on the larval biotope than on the season or the larvicidal application. More bacilli were found in soil (10(4)-10(6) spores/g) than on plant samples (10(2)-10(4) spores/g). The abundance in water (10(5) to 10(7) spores/L) appeared to be correlated to the water level of the breeding site. The number of Bti spores increased just after application, after declining; no spores were detected in soil or water 3 months after application. However, low numbers of Bti spores were present on foliage from three of the four studied plant strata. In conclusion, the larvicidal application has very little impact on Bacillus spp. flora after one breeding season (two applications).

  9. Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spore DNA

    International Nuclear Information System (INIS)

    Setlow, B.; Hand, A.R.; Setlow, P.

    1991-01-01

    Small, acid-soluble proteins (SASP) of the alpha/beta-type are associated with DNA in spores of Bacillus subtilis. Induction of synthesis of alpha/beta-type SASP in Escherichia coli resulted in rapid cessation of DNA synthesis, followed by a halt in RNA and then protein accumulation, although significant mRNA and protein synthesis continued. There was a significant loss in viability associated with SASP synthesis in E. coli: recA+ cells became extremely long filaments, whereas recA mutant cells became less filamentous. The nucleoids of cells with alpha/beta-type SASP were extremely condensed, as viewed in both light and electron microscopes, and immunoelectron microscopy showed that the alpha/beta-type SASP were associated with the cell DNA. Induction of alpha/beta-type SASP synthesis in E. coli increased the negative superhelical density of plasmid DNA by approximately 20%; UV irradiation of E. coli with alpha/beta-type SASP gave reduced yields of thymine dimers but significant amounts of the spore photoproduct. These changes in E. coli DNA topology and photochemistry due to alpha/beta-type SASP are similar to the effects of alpha/beta-type SASP on the DNA in Bacillus spores, further suggesting that alpha/beta-type SASP are a major factor determining DNA properties in bacterial spores

  10. 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

  11. Test methods and response surface models for hot, humid air decontamination of materials contaminated with dirty spores of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam.

    Science.gov (United States)

    Buhr, T L; Young, A A; Barnette, H K; Minter, Z A; Kennihan, N L; Johnson, C A; Bohmke, M D; DePaola, M; Cora-Laó, M; Page, M A

    2015-11-01

    To develop test methods and evaluate survival of Bacillus anthracis ∆Sterne or Bacillus thuringiensis Al Hakam on materials contaminated with dirty spore preparations after exposure to hot, humid air using response surface modelling. Spores (>7 log10 ) were mixed with humic acid + spent sporulation medium (organic debris) or kaolin (dirt debris). Spore samples were then dried on five different test materials (wiring insulation, aircraft performance coating, anti-skid, polypropylene, and nylon). Inoculated materials were tested with 19 test combinations of temperature (55, 65, 75°C), relative humidity (70, 80, 90%) and time (1, 2, 3 days). The slowest spore inactivation kinetics was on nylon webbing and/or after addition of organic debris. Hot, humid air effectively decontaminates materials contaminated with dirty Bacillus spore preparations; debris and material interactions create complex decontamination kinetic patterns; and B. thuringiensis Al Hakam is a realistic surrogate for B. anthracis. Response surface models of hot, humid air decontamination were developed which may be used to select decontamination parameters for contamination scenarios including aircraft. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

  12. 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

  13. Determining the Role of Multicopper Oxidases in Manganese(II) Oxidation by Marine Bacillus Spores

    Science.gov (United States)

    Dick, G. J.; Tebo, B. M.

    2005-12-01

    Bacteria play an important role in the environmental cycling of Mn by oxidizing soluble Mn(II) and forming insoluble Mn(III/IV) oxides. These biogenic Mn oxides are renowned for their strong sorptive and oxidative properties, which control the speciation and availability of many metals and organic compounds. A wide variety of bacteria are known to catalyze the oxidation of Mn(II); one of the most frequently isolated types are Bacillus species that oxidize Mn(II) only as metabolically dormant spores. We are using genetic and biochemical methods to study the molecular mechanisms of this process in these organisms. mnxG, a gene related to the multicopper oxidase (MCO) family of enzymes, is required for Mn(II) oxidation in the model organism, Bacillus sp. strain SG-1. Mn(II)-oxidizing activity can be detected in crude protein extracts of the exosporium and as a discrete band in SDS-PAGE gels, however previous attempts to purify or identify this Mn(II)-oxidizing enzyme have failed. A direct link between the Mn(II)-oxidizing enzyme and the MCO gene suspected to encode it has never been made. We used genetic and biochemical methods to investigate the role of the MCO in the mechanism of Mn(II) oxidation. Comparative analysis of the mnx operon from several diverse Mn(II)-oxidizing Bacillus spores revealed that mnxG is the most highly conserved gene in the operon, and that copper binding sites are highly conserved. As with Mn(II) oxidases from other organisms, heterologous expression of the Bacillus mnxG in E. coli did not yield an active Mn(II) oxidase. Purifying sufficient quantities of the native Mn(II) oxidase from Bacillus species for biochemical characterization has proven difficult because the enzyme does not appear to be abundant, and it is highly insoluble. We were able to partially purify the Mn(II) oxidase, and to analyze the active band by in-gel trypsin digestion followed by tandem mass spectrometry (MS/MS). MS/MS spectra provided a conclusive match to mnx

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

    Science.gov (United States)

    2008-03-01

    gentlemen are approachable and extremely well versed in their fields. Dr. Burggraf’s passion for learning coupled with the patience necessary to allow...wide spread employment on civilian targets has increased. Indeed, the well known biological agent Anthrax, Bacillus anthracis (B.a.), was recently...reached at 0.5 seconds with the heat from the fireball lasting a total of 4 seconds ( Orson , J.A. 2003; 104). From this data, the short time-temperature

  15. The Cooperative and Interdependent Roles of GerA, GerK, and Ynd in Germination of Bacillus licheniformis Spores.

    Science.gov (United States)

    Borch-Pedersen, Kristina; Lindbäck, Toril; Madslien, Elisabeth H; Kidd, Shani W; O'Sullivan, Kristin; Granum, Per Einar; Aspholm, Marina

    2016-07-15

    When nutrients are scarce, Bacillus species form metabolically dormant and extremely resistant spores that enable survival over long periods of time under conditions not permitting growth. The presence of specific nutrients triggers spore germination through interaction with germinant receptors located in the spore's inner membrane. Bacillus licheniformis is a biotechnologically important species, but it is also associated with food spoilage and food-borne disease. The B. licheniformis ATCC 14580/DSM13 genome exhibits three gerA family operons (gerA, gerK, and ynd) encoding germinant receptors. We show that spores of B. licheniformis germinate efficiently in response to a range of different single l-amino acid germinants, in addition to a weak germination response seen with d-glucose. Mutational analyses revealed that the GerA and Ynd germination receptors function cooperatively in triggering an efficient germination response with single l-amino acid germinants, whereas the GerK germination receptor is essential for germination with d-glucose. Mutant spores expressing only GerA and GerK or only Ynd and GerK show reduced or severely impaired germination responses, respectively, with single l-amino acid germinants. Neither GerA nor Ynd could function alone in stimulating spore germination. Together, these results functionally characterize the germination receptor operons present in B. licheniformis We demonstrate the overlapping germinant recognition patterns of the GerA and Ynd germination receptors and the cooperative functionalities between GerA, Ynd, and GerK in inducing germination. To ensure safe food production and durable foods, there is an obvious need for more knowledge on spore-forming bacteria. It is the process of spore germination that ultimately leads to food spoilage and food poisoning. Bacillus licheniformis is a biotechnologically important species that is also associated with food spoilage and food-borne disease. Despite its importance, the

  16. Analysis of the synergistic effect of radiation and heat on bacteriophage T4 and spores Bacillus subtilis

    International Nuclear Information System (INIS)

    Komarov, V.P.; Petin, V.G.

    1987-01-01

    Experimental data are interpreted in terms of a half-empiric model of synergism obtained for bacteriophage T4 and Bacillus subtilis spores exposed to ionizing radiation of different dose rates at elevated temperatures. The model permits to optimize the ratio of both factors for effective sterilization

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

    NARCIS (Netherlands)

    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

  18. 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.

  19. 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.

  20. Heat resistance of Bacillus cereus spores: effects of milk constituents and stabilizing additives.

    Science.gov (United States)

    Mazas, M; López, M; Martínez, S; Bernardo, A; Martin, R

    1999-04-01

    Heat resistance of Bacillus cereus spores (ATCC 7004, 4342, and 9818) heated in different types of milk (skim, whole, and concentrated skim milk), skim milk containing stabilizing additives (sodium citrate, monopotassium phosphate, or disodium phosphate, 0.1%), and cream was investigated. Thermal resistance experiments were performed at temperatures within the range of 92 to 115 degrees C under continuous monitoring of pH. For strain 4342 no significant differences (P < 0.05) in D values were detected in any case. For strains 7004 and 9818 higher D values of about 20% were obtained in whole and concentrated skim milk than those calculated in skim milk. From all stabilizing additives tested, only sodium citrate and sodium phosphate increased the heat resistance for strain 9818. However, when the menstruum pH was measured at the treatment temperature, different pH values were found between the heating media. The differences in heat resistance observed could be due to a pH effect rather than to the difference in the substrates in which spores were heated. In contrast, when cream (fat content 20%) was used, lower D values were obtained, especially for strains 7004 and 9818. z values were not significantly modified by the milk composition, with an average z value of 7.95+/-0.20 degrees C for strain 7004, 7.88+/-0.10 degrees C for strain 4342, and 9.13+/-0.16 degrees C for strain 9818.

  1. Dynamic Predictive Model for Growth of Bacillus cereus from Spores in Cooked Beans.

    Science.gov (United States)

    Juneja, Vijay K; Mishra, Abhinav; Pradhan, Abani K

    2018-02-01

    Kinetic growth data for Bacillus cereus grown from spores were collected in cooked beans under several isothermal conditions (10 to 49°C). Samples were inoculated with approximately 2 log CFU/g heat-shocked (80°C for 10 min) spores and stored at isothermal temperatures. B. cereus populations were determined at appropriate intervals by plating on mannitol-egg yolk-polymyxin agar and incubating at 30°C for 24 h. Data were fitted into Baranyi, Huang, modified Gompertz, and three-phase linear primary growth models. All four models were fitted to the experimental growth data collected at 13 to 46°C. Performances of these models were evaluated based on accuracy and bias factors, the coefficient of determination ( R 2 ), and the root mean square error. Based on these criteria, the Baranyi model best described the growth data, followed by the Huang, modified Gompertz, and three-phase linear models. The maximum growth rates of each primary model were fitted as a function of temperature using the modified Ratkowsky model. The high R 2 values (0.95 to 0.98) indicate that the modified Ratkowsky model can be used to describe the effect of temperature on the growth rates for all four primary models. The acceptable prediction zone (APZ) approach also was used for validation of the model with observed data collected during single and two-step dynamic cooling temperature protocols. When the predictions using the Baranyi model were compared with the observed data using the APZ analysis, all 24 observations for the exponential single rate cooling were within the APZ, which was set between -0.5 and 1 log CFU/g; 26 of 28 predictions for the two-step cooling profiles also were within the APZ limits. The developed dynamic model can be used to predict potential B. cereus growth from spores in beans under various temperature conditions or during extended chilling of cooked beans.

  2. 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.

    Science.gov (United States)

    Correa, Elon; Goodacre, Royston

    2011-01-26

    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. 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. 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.

  3. 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.

  4. Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis

    Science.gov (United States)

    Ramírez-Guadiana, Fernando H.; Barraza-Salas, Marcelo; Ramírez-Ramírez, Norma; Ortiz-Cortés, Mayte; Setlow, Peter

    2012-01-01

    The nucleotide excision repair (NER) and spore photoproduct lyase DNA repair pathways are major determinants of Bacillus subtilis spore resistance to UV radiation. We report here that a putative ultraviolet (UV) damage endonuclease encoded by ywjD confers protection to developing and dormant spores of B. subtilis against UV DNA damage. In agreement with its predicted function, a His6-YwjD recombinant protein catalyzed the specific incision of UV-irradiated DNA in vitro. The maximum expression of a reporter gene fusion to the ywjD opening reading frame occurred late in sporulation, and this maximal expression was dependent on the forespore-specific RNA polymerase sigma factor, σG. Although the absence of YwjD and/or UvrA, an essential protein of the NER pathway, sensitized developing spores to UV-C, this effect was lower when these cells were treated with UV-B. In contrast, UV-B but not UV-C radiation dramatically decreased the survival of dormant spores deficient in both YwjD and UvrA. The distinct range of lesions generated by UV-C and UV-B and the different DNA photochemistry in developing and dormant spores may cause these differences. We postulate that in addition to the UvrABC repair system, developing and dormant spores of B. subtilis also rely on an alternative excision repair pathway involving YwjD to deal with the deleterious effects of various UV photoproducts. PMID:22961846

  5. Spectroscopy and viability of Bacillus subtilis spores after ultraviolet irradiation: implications for the detection of potential bacterial life on Europa.

    Science.gov (United States)

    Noell, Aaron C; Ely, Tucker; Bolser, Diana K; Darrach, Halley; Hodyss, Robert; Johnson, Paul V; Hein, Jeffrey D; Ponce, Adrian

    2015-01-01

    One of the most habitable environments in the Solar System outside of Earth may exist underneath the ice on Europa. In the near future, our best chance to look for chemical signatures of a habitable environment (or life itself) will likely be at the inhospitable icy surface. Therefore, it is important to understand the ability of organic signatures of life and life itself to persist under simulated europan surface conditions. Toward that end, this work examined the UV photolysis of Bacillus subtilis spores and their chemical marker dipicolinic acid (DPA) at temperatures and pressures relevant to Europa. In addition, inactivation curves for the spores at 100 K, 100 K covered in one micron of ice, and 298 K were measured to determine the probability for spore survival at the surface. Fourier transform infrared spectra of irradiated DPA showed a loss of carboxyl groups to CO2 as expected but unexpectedly showed significant opening of the heterocyclic ring, even for wavelengths>200 nm. Both DPA and B. subtilis spores showed identical unknown spectral bands of photoproducts after irradiation, further highlighting the importance of DPA in the photochemistry of spores. Spore survival was enhanced at 100 K by ∼5× relative to 298 K, but 99.9% of spores were still inactivated after the equivalent of ∼25 h of exposure on the europan surface.

  6. Rapid optimization of spore production from Bacillus amyloliquefaciens in submerged cultures based on dipicolinic acid fluorimetry assay.

    Science.gov (United States)

    Ren, Hang; Su, Ya-Ting; Guo, Xiao-Hua

    2018-02-16

    Some optimization techniques have been widely applied for spore fermentation based on the plate counting. This study optimized the culture medium for the spore production of Bacillus amyloliquefaciens BS-20 and investigated the feasibility of using a dipicolonic acid (DPA) fluorimetry assay as a simpler alternative to plate counting for evaluating spore yields. Through the single-factor experiment, the metal ions and agro-industrial raw materials that significantly enhanced spore production were determined. After conducting a response surface methodology (RSM) analysis of several metal ions, the combined use of optimum concentrations of Mn 2+ , Fe 2+ , and Ca 2+ in culture media produced a 3.4-fold increase in spore yields. Subsequently, supplementing soybean meal and corn meal with optimum concentrations determined by another RSM analysis produced an 8.8-fold increase. The final spore concentration from a culture medium incorporating optimum concentrations of the metal ions and raw materials mentioned above was verified to reach (8.05 ± 0.70) × 10 9  CFU/mL by both DPA fluorimetry and plate counting. The results suggest that the use of DPA fluorescence intensity as an alternative value to colony counting provides a general method for assessing spore yields with less work and shorter time.

  7. Hot, humid air decontamination of a C-130 aircraft contaminated with spores of two acrystalliferous Bacillus thuringiensis strains, surrogates for Bacillus anthracis.

    Science.gov (United States)

    Buhr, T L; Young, A A; Bensman, M; Minter, Z A; Kennihan, N L; Johnson, C A; Bohmke, M D; Borgers-Klonkowski, E; Osborn, E B; Avila, S D; Theys, A M G; Jackson, P J

    2016-04-01

    To develop test methods and evaluate survival of Bacillus thuringiensis kurstaki cry(-) HD-1 and B. thuringiensis Al Hakam spores after exposure to hot, humid air inside of a C-130 aircraft. Bacillus thuringiensis spores were either pre-inoculated on 1 × 2 or 2 × 2 cm substrates or aerosolized inside the cargo hold of a C-130 and allowed to dry. Dirty, complex surfaces (10 × 10 cm) swabbed after spore dispersal showed a deposition of 8-10 log10 m(-2) through the entire cargo hold. After hot, humid air decontamination at 75-80°C, 70-90% relative humidity for 7 days, 87 of 98 test swabs covering 0·98 m(2) , showed complete spore inactivation. There was a total of 1·67 log10 live CFU detected in 11 of the test swabs. Spore inactivation in the 98 test swabs was measured at 7·06 log10 m(-2) . Laboratory test methods for hot, humid air decontamination were scaled for a large-scale aircraft field test. The C-130 field test demonstrated that hot, humid air can be successfully used to decontaminate an aircraft. Transition of a new technology from research and development to acquisition at a Technology Readiness Level 7 is unprecedented. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  8. Marine Bacillus spores as catalysts for oxidative precipitation and sorption of metals.

    Science.gov (United States)

    Francis, C A; Tebo, B M

    1999-08-01

    The oxidation of soluble manganese(II) to insoluble Mn(III,IV) oxide precipitates plays an important role in the environment. These Mn oxides are known to oxidize numerous organic and inorganic compounds, scavenge a variety of other metals on their highly charged surfaces, and serve as electron acceptors for anaerobic respiration. Although the oxidation of Mn(II) in most environments is believed to be bacterially-mediated, the underlying mechanisms of catalysis are not well understood. In recent years, however, the application of molecular biological approaches has provided new insights into these mechanisms. Genes involved in Mn oxidation were first identified in our model organism, the marine Bacillus sp. strain SG-1, and subsequently have been identified in two other phylogenetically distinct organisms, Leptothrix discophora and Pseudomonas putida. In all three cases, enzymes related to multicopper oxidases appear to be involved, suggesting that copper may play a universal role in Mn(II) oxidation. In addition to catalyzing an environmentally important process, organisms capable of Mn(II) oxidation are potential candidates for the removal, detoxification, and recovery of metals from the environment. The Mn(II)-oxidizing spores of the marine Bacillus sp. strain SG-1 show particular promise, due to their inherent physically tough nature and unique capacity to bind and oxidatively precipitate metals without having to sustain growth.

  9. Modeling the recovery of heat-treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 spores at suboptimal temperature and pH using growth limits.

    Science.gov (United States)

    Trunet, C; Mtimet, N; Mathot, A-G; Postollec, F; Leguerinel, I; Sohier, D; Couvert, O; Carlin, F; Coroller, L

    2015-01-01

    The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. Recovery of heat treated Bacillus cereus spores is affected by matrix composition and factors with putative functions in damage repair

    Directory of Open Access Journals (Sweden)

    Alicja Katarzyna Warda

    2016-07-01

    Full Text Available The ability of spores to recover and grow out after food processing is affected by cellular factors and by the outgrowth conditions. In the current communication we studied the recovery and outgrowth of individually sorted spores in BHI and rice broth media and on agar plates using flow cytometry (FCM. We show that recovery of wet heat treated Bacillus cereus ATCC 14579 spores is affected by matrix composition with highest recovery in BHI broth or on rice agar plates, compared to BHI agar plates and rice broth. Data show that not only media composition but also its liquid or solid state affect the recovery of heat treated spores. To determine the impact of factors with putative roles in recovery of heat treated spores, specific genes previously shown to be highly expressed in outgrowing heat-treated spores were selected for mutant construction. Spores of nine B. cereus ATCC 14579 deletion mutants were obtained and their recovery from wet heat treatment was evaluated using BHI and rice broth and agar plates. Deletion mutant spores showed different capacity to recover from heat treatment compared to wild type with the most pronounced effect for a mutant lacking BC5242, a gene encoding a membrane protein with C2C2 zinc finger which resulted in over 95% reduction in recovery compared to the wild type in BHI broth. Notably, similar relative performance of wild type and mutants was observed using the other recovery conditions.We obtained insights on the impact of matrix composition and state on recovery of individually sorted heat treated spores and identified cellular factors with putative roles in this process. These results may provide leads for future developments in design of more efficient combined preservation treatments.

  11. Recovery of Heat Treated Bacillus cereus Spores Is Affected by Matrix Composition and Factors with Putative Functions in Damage Repair.

    Science.gov (United States)

    Warda, Alicja K; Tempelaars, Marcel H; Abee, Tjakko; Nierop Groot, Masja N

    2016-01-01

    The ability of spores to recover and grow out after food processing is affected by cellular factors and by the outgrowth conditions. In the current communication we studied the recovery and outgrowth of individually sorted spores in BHI and rice broth media and on agar plates using flow cytometry. We show that recovery of wet heat treated Bacillus cereus ATCC 14579 spores is affected by matrix composition with highest recovery in BHI broth or on rice agar plates, compared to BHI agar plates and rice broth. Data show that not only media composition but also its liquid or solid state affect the recovery of heat treated spores. To determine the impact of factors with putative roles in recovery of heat treated spores, specific genes previously shown to be highly expressed in outgrowing heat-treated spores were selected for mutant construction. Spores of nine B. cereus ATCC 14579 deletion mutants were obtained and their recovery from wet heat treatment was evaluated using BHI and rice broth and agar plates. Deletion mutant spores showed different capacity to recover from heat treatment compared to wild type with the most pronounced effect for a mutant lacking BC5242, a gene encoding a membrane protein with C2C2 zinc finger which resulted in over 95% reduction in recovery compared to the wild type in BHI broth. Notably, similar relative performance of wild type and mutants was observed using the other recovery conditions. We obtained insights on the impact of matrix composition and state on recovery of individually sorted heat treated spores and identified cellular factors with putative roles in this process. These results may provide leads for future developments in design of more efficient combined preservation treatments.

  12. 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.,...

  13. Proteins YlaJ and YhcN contribute to the efficiency of spore germination in Bacillus subtilis.

    Science.gov (United States)

    Johnson, Christian L; Moir, Anne

    2017-04-01

    The YlaJ and YhcN spore lipoproteins of Bacillus subtilis contain a common domain, and are of unknown function. Homologues of YlaJ or YhcN are widespread in Bacilli and are also encoded in those Clostridia that use cortex lytic enzymes SleB and CwlJ for cortex hydrolysis during germination. In B. subtilis, we report that single and double mutants lacking YlaJ and/or YhcN show a reduced rate of spore germination in L-alanine, with a delay in loss of heat resistance, release of dipicolinic acid and OD fall. If B. subtilis spores lack the cortex lytic enzyme CwlJ, spore cortex degradation and subsequent outgrowth to form colonies is strictly dependent on the other cortex lytic enzyme SleB, allowing a test of SleB function; in a cwlJ mutant background, the combined loss of both ylaJ and yhcN genes resulted in a spore population in which only 20% of spores germinated and outgrew to form colonies, suggesting that SleB activity is compromised. YlaJ and YhcN have a role in germination that is not yet well defined, but these proteins are likely to contribute, directly or indirectly, to early events in germination, including effective SleB function. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. 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)

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

    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

  15. Responses to accelerated heavy ions of spores of Bacillus subtilis of different repair capacity

    International Nuclear Information System (INIS)

    Baltschukat, K.; Horneck, G.

    1991-01-01

    Inactivation, mutagenesis of histidine reversion and the involvement of DNA repair were studied in spores of Bacillus subtilis irradiated with heavy ions at LBL, Berkeley and GSI, Darmstadt. Five groups of ions (from boron to uranium) were used with residual energies from 0.2 MeV/u up to 18.6 MeV/u; in addition, carbon ions were used with a residual energy of 120 MeV/u. Action cross sections of both inactivation and mutagenesis show a similar dependence on ion mass and energy: For lighter ions (Z≤10), the lethal response is nearly energy independent (Z=10) or decreasing with energy (Z≤6); these light ions, up to 18.6 MeV/u, induce hardly any mutations. For heavier ions (Z≥26), the lethal as well as the mutagenic responses increase with ion mass and energy up to a maximum or saturation. The efficiency of DNA repair to improve survival and the mutagenic efficiency per lethal event, both, increase with ion energy up to a saturation value which, depending on strain and endpoint, either roughtly coincides with the X-ray value or is smaller than that after X-ray treatment. For repair based on recombination events, the increase in the survival effects with ion energy is more pronounced than for that based on repair replication. At energies of 1 MeV/u or below, neither DNA repair nor mutation induction appear to be significant. The results support previous suggestions on the importance of the radial distribution of the energy around the ion track in biological action cross section and the evidence that the entire core of the spore represents the sensitive site in responses to heavy ions. (orig.)

  16. Comparative evaluation of DNA extraction methods for amplification by qPCR of superficial vs intracellular DNA from Bacillus spores.

    Science.gov (United States)

    Brauge, Thomas; Faille, Christine; Inglebert, Gaëlle; Dubois, Thomas; Morieux, Paul; Slomianny, Christian; Midelet-Bourdin, Graziella

    2018-02-02

    This study was designed to assess the efficiency of eight extraction methods regarding their ability to release superficial (exogenous) and intracellular (endogenous) DNA from B. cereus spores for subsequent analysis by quantitative PCR (qPCR). B. cereus spore suspensions were subjected to both commercial DNA extraction kits and mechanical DNA extraction methods. The spores were observed by transmission electron microscopy to evaluate any damage caused during extraction. The efficiency of both extraction and purification were assessed using a qPCR assay targeting the bclA gene. Most of the extraction methods assessed, except the passage through the French press or the use of the QIAamp DNA Blood Mini kit without 95°C treatment, allowed the amplification of significant amounts of DNA. By using propidium monoazide, which is a photoreactive DNA-binding dye, the presence of non-negligible amounts of amplifiable DNA at the spore surface was highlighted. A further set of extraction assays was then performed on spores previously treated with PMA. The results of this study show that both superficial and intracellular spore DNA can be released by extraction methods to a greater or lesser extent and then further amplified by qPCR. The Precellys extraction allowed the detection of both intracellular and superficial DNA, the DNeasy Blood & Tissue kit the specific detection of intracellular DNA, while the Instagene kit detected only superficial DNA. Of the methods tested in this study, the Precellys extraction was the most efficient in terms of further DNA detection. In order to verify the presence or absence of B. cereus spores in food or on surfaces in the food environment, the use of an efficient extraction method is required, followed by a qPCR analysis on the DNA released. Conversely, in order to quantify the population of Bacillus spores, any superficial DNA must be blocked, e.g. with PMA, prior to intracellular DNA extraction and further amplification. Copyright © 2018

  17. 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

    processes also have significant applied interest, since (i) spores are major agents of food spoilage and food poisoning and (ii) spores’ extreme...by U N IV O F C O N N E C T IC U T http://aem .asm .org/ D ow nloaded from needed in conjunction with HP to inactivate bacterial spores and...achieve the commercial sterility of low-acid foods (22). While HP is probably most often used as a single treatment, a number of studies have demonstrated

  18. Bacillus nealsonii sp. nov., isolated from a spacecraft-assembly facility, whose spores are gamma-radiation resistant

    Science.gov (United States)

    Venkateswaran, Kasthuri; Kempf, Michael; Chen, Fei; Satomi, Masataka; Nicholson, Wayne; Kern, Roger

    2003-01-01

    One of the spore-formers isolated from a spacecraft-assembly facility, belonging to the genus Bacillus, is described on the basis of phenotypic characterization, 16S rDNA sequence analysis and DNA-DNA hybridization studies. It is a Gram-positive, facultatively anaerobic, rod-shaped eubacterium that produces endospores. The spores of this novel bacterial species exhibited resistance to UV, gamma-radiation, H2O2 and desiccation. The 18S rDNA sequence analysis revealed a clear affiliation between this strain and members of the low G+C Firmicutes. High 16S rDNA sequence similarity values were found with members of the genus Bacillus and this was supported by fatty acid profiles. The 16S rDNA sequence similarity between strain FO-92T and Bacillus benzoevorans DSM 5391T was very high. However, molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in this genus, but DNA-DNA hybridization data support the proposal of FO-92T as Bacillus nealsonii sp. nov. (type strain is FO-92T =ATCC BAAM-519T =DSM 15077T).

  19. The Prevalence and Control of Bacillus and Related Spore-Forming Bacteria in the Dairy Industry

    Science.gov (United States)

    Gopal, Nidhi; Hill, Colin; Ross, Paul R.; Beresford, Tom P.; Fenelon, Mark A.; Cotter, Paul D.

    2015-01-01

    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 pasteurization 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. PMID:26733963

  20. 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.

  1. Effective Thermal Inactivation of the Spores of Bacillus cereus Biofilms Using Microwave.

    Science.gov (United States)

    Park, Hyong Seok; Yang, Jungwoo; Choi, Hee Jung; Kim, Kyoung Heon

    2017-07-28

    Microwave sterilization was performed to inactivate the spores of biofilms of Bacillus cereus involved in foodborne illness. The sterilization conditions, such as the amount of water and the operating temperature and treatment time, were optimized using statistical analysis based on 15 runs of experimental results designed by the Box-Behnken method. Statistical analysis showed that the optimal conditions for the inactivation of B. cereus biofilms were 14 ml of water, 108°C of temperature, and 15 min of treatment time. Interestingly, response surface plots showed that the amount of water is the most important factor for microwave sterilization under the present conditions. Complete inactivation by microwaves was achieved in 5 min, and the inactivation efficiency by microwave was obviously higher than that by conventional steam autoclave. Finally, confocal laser scanning microscopy images showed that the principal effect of microwave treatment was cell membrane disruption. Thus, this study can contribute to the development of a process to control food-associated pathogens.

  2. 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. Copyright (c) 2007 John Wiley & Sons, Ltd.

  3. Impacts of sporulation temperature, exposure to compost matrix and temperature on survival of Bacillus cereus spores during livestock mortality composting.

    Science.gov (United States)

    Stanford, K; Reuter, T; Gilroyed, B H; McAllister, T A

    2015-04-01

    To investigate impact of sporulation and compost temperatures on feasibility of composting for disposal of carcasses contaminated with Bacillus anthracis. Two strains of B. cereus, 805 and 1391, were sporulated at either 20 or 37°C (Sporulation temperature, ST) and 7 Log10 CFU g(-1) spores added to autoclaved manure in nylon bags (pore size 50 μm) or in sealed vials. Vials and nylon bags were embedded into compost in either a sawdust or manure matrix each containing 16 bovine mortalities (average weight 617 ± 33 kg), retrieved from compost at intervals over 217 days and survival of B. cereus spores assessed. A ST of 20°C decreased spore survival by 1·4 log10 CFU g(-1) (P Compost temperatures >55°C reduced spore survival (P compost temperatures were key factors influencing survival of B. cereus spores in mortality compost. Composting may be most appropriate for the disposal of carcasses infected with B. anthracis at ambient temperatures ≤20°C under thermophillic composting conditions (>55°C). © 2015 The Society for Applied Microbiology.

  4. Role played by exosporium glycoproteins in the surface properties of Bacillus cereus spores and in their adhesion to stainless steel.

    Science.gov (United States)

    Lequette, Yannick; Garénaux, Estelle; Tauveron, Grégoire; Dumez, Sylvain; Perchat, Stéphane; Slomianny, Christian; Lereclus, Didier; Guérardel, Yann; Faille, Christine

    2011-07-01

    Bacillus cereus spores are surrounded by a loose-fitting layer called the exosporium, whose distal part is mainly formed from glycoproteins. The role played by the exosporium glycoproteins of B. cereus ATCC 14579 (BclA and ExsH) was investigated by considering hydrophobicity and charge, as well as the properties of spore adhesion to stainless steel. The absence of BclA increased both the isoelectric point (IEP) and hydrophobicity of whole spores while simultaneously reducing the interaction between spores and stainless steel. However, neither the hydrophobicity nor the charge associated with BclA could explain the differences in the adhesion properties. Conversely, ExsH, another exosporium glycoprotein, did not play a significant role in spore surface properties. The monosaccharide analysis of B. cereus ATCC 14579 showed different glycosylation patterns on ExsH and BclA. Moreover, two specific glycosyl residues, namely, 2-O-methyl-rhamnose (2-Me-Rha) and 2,4-O-methyl-rhamnose (2,4-Me-Rha), were attached to BclA, in addition to the glycosyl residues already reported in B. anthracis.

  5. Development of an eco-friendly approach for biogenesis of silver nanoparticles using spores of Bacillus athrophaeus.

    Science.gov (United States)

    Hosseini-Abari, Afrouzossadat; Emtiazi, Giti; Ghasemi, Seyed Mahdi

    2013-12-01

    The biological synthesis methods have been emerging as a promising new approach for production of nanoparticles due to their simplicity and non-toxicity. In the present study, spores of Bacillus athrophaeus were used to achieve the objective of developing a green synthesis method of silver nanoparticles. Enzyme assay revealed that the spores and their heat inactivated forms (microcapsules) were highly active and their enzymatic contents differed from the vegetative cells. Laccase, glucose oxidase, and alkaline phosphatase activities were detected in the dormant forms, but not in the vegetative cells. Although no nanoparticle was produced by active cells of B. athrophaeus, both spores and microcapsules were efficiently capable of reducing the silver ions (Ag⁺) to elemental silver (Ag⁰) leading to the formation of nanoparticles from silver nitrate (AgNO₃). The presence of biologically synthesized silver nanoparticles was determined by obtaining broad spectra with maximum absorbance at 400 nm in UV-visible spectroscopy. The X-ray diffraction analysis pattern revealed that the nanoscale particles have crystalline nature with various topologies, as confirmed by transmission electron microscopy (TEM). The TEM micrograph showed the nanocrystal structures with dimensions ranging from 5 to 30 nm. Accordingly, the spore mixture could be employed as a factory for detoxification of heavy metals and subsequent production of nanoparticles. This research introduces an environmental friendly and cost effective biotechnological process for the extracellular synthesis of silver nanoparticles using the bacterial spores.

  6. 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

  7. 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Assessing the Impact of Germination and Sporulation Conditions on the Adhesion of Bacillus Spores to Glass and Stainless Steel by Fluid Dynamic Gauging.

    Science.gov (United States)

    Xu Zhou, Ke; Li, Nan; Christie, Graham; Wilson, D Ian

    2017-11-01

    The adhesion of spores of 3 Bacillus species with distinctive morphologies to stainless steel and borosilicate glass was studied using the fluid dynamic gauging technique. Marked differences were observed between different species of spores, and also between spores of the same species prepared under different sporulation conditions. Spores of the food-borne pathogen B. cereus were demonstrated to be capable of withstanding shear stresses greater than 1500 Pa when adhered to stainless steel, in contrast to spores of Bacillus subtilis and Bacillus megaterium, which detached in response to lower shear stress. An extended DLVO model was shown to be capable of predicting the relative differences in spore adhesion between spores of different species and different culture conditions, but did not predict absolute values of force of adhesion well. Applying the model to germinating spores showed a significant reduction in adhesion force shortly after triggering germination, indicating a potential strategy to achieve enhanced removal of spores from surfaces in response to shear stress, such as during cleaning-in-place procedures. Spore-forming bacteria are a concern to the food industry because they have the potential to cause food-borne illness and product spoilage, while being strongly adhesive to processing surfaces and resistant to cleaning-in-place procedures. This work is of significance to the food processors and manufacturers because it offers insight to the properties of spore adhesion and identifies a potential strategy to facilitate the removal of spores during cleaning procedures. © 2017 The Authors. Journal of Food Science published by Wiley Periodicals, Inc. on behalf of Institute of Food Technologists.

  9. Developmentally-regulated excision of the SPβ prophage reconstitutes a gene required for spore envelope maturation in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Kimihiro Abe

    2014-10-01

    Full Text Available Temperate phages infect bacteria by injecting their DNA into bacterial cells, where it becomes incorporated into the host genome as a prophage. In the genome of Bacillus subtilis 168, an active prophage, SPβ, is inserted into a polysaccharide synthesis gene, spsM. Here, we show that a rearrangement occurs during sporulation to reconstitute a functional composite spsM gene by precise excision of SPβ from the chromosome. SPβ excision requires a putative site-specific recombinase, SprA, and an accessory protein, SprB. A minimized SPβ, where all the SPβ genes were deleted, except sprA and sprB, retained the SPβ excision activity during sporulation, demonstrating that sprA and sprB are necessary and sufficient for the excision. While expression of sprA was observed during vegetative growth, sprB was induced during sporulation and upon mitomycin C treatment, which triggers the phage lytic cycle. We also demonstrated that overexpression of sprB (but not of sprA resulted in SPβ prophage excision without triggering the lytic cycle. These results suggest that sprB is the factor that controls the timing of phage excision. Furthermore, we provide evidence that spsM is essential for the addition of polysaccharides to the spore envelope. The presence of polysaccharides on the spore surface renders the spore hydrophilic in water. This property may be beneficial in allowing spores to disperse in natural environments via water flow. A similar rearrangement occurs in Bacillus amyloliquefaciens FZB42, where a SPβ-like element is excised during sporulation to reconstitute a polysaccharide synthesis gene, suggesting that this type of gene rearrangement is common in spore-forming bacteria because it can be spread by phage infection.

  10. Developmentally-Regulated Excision of the SPβ Prophage Reconstitutes a Gene Required for Spore Envelope Maturation in Bacillus subtilis

    Science.gov (United States)

    Abe, Kimihiro; Kawano, Yuta; Iwamoto, Keito; Arai, Kenji; Maruyama, Yuki; Eichenberger, Patrick; Sato, Tsutomu

    2014-01-01

    Temperate phages infect bacteria by injecting their DNA into bacterial cells, where it becomes incorporated into the host genome as a prophage. In the genome of Bacillus subtilis 168, an active prophage, SPβ, is inserted into a polysaccharide synthesis gene, spsM. Here, we show that a rearrangement occurs during sporulation to reconstitute a functional composite spsM gene by precise excision of SPβ from the chromosome. SPβ excision requires a putative site-specific recombinase, SprA, and an accessory protein, SprB. A minimized SPβ, where all the SPβ genes were deleted, except sprA and sprB, retained the SPβ excision activity during sporulation, demonstrating that sprA and sprB are necessary and sufficient for the excision. While expression of sprA was observed during vegetative growth, sprB was induced during sporulation and upon mitomycin C treatment, which triggers the phage lytic cycle. We also demonstrated that overexpression of sprB (but not of sprA) resulted in SPβ prophage excision without triggering the lytic cycle. These results suggest that sprB is the factor that controls the timing of phage excision. Furthermore, we provide evidence that spsM is essential for the addition of polysaccharides to the spore envelope. The presence of polysaccharides on the spore surface renders the spore hydrophilic in water. This property may be beneficial in allowing spores to disperse in natural environments via water flow. A similar rearrangement occurs in Bacillus amyloliquefaciens FZB42, where a SPβ-like element is excised during sporulation to reconstitute a polysaccharide synthesis gene, suggesting that this type of gene rearrangement is common in spore-forming bacteria because it can be spread by phage infection. PMID:25299644

  11. 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.

  12. Response surface modeling for the inactivation of Bacillus subtilis subsp. niger spores by chlorine dioxide gas in an enclosed space.

    Science.gov (United States)

    Wang, Tao; Qi, Jiancheng; Wu, Jinhui; Hao, Limei; Yi, Ying; Lin, Song; Zhang, Zongxing

    2016-05-01

    Bacillus subtilis subsp. niger spores are a commonly used biological indicator to evaluate the disinfection of an enclosed space. In the present study, chlorine dioxide (ClO2) gas was applied to inactivate B. subtilis subsp. niger spores in an enclosed space. The effects of the ClO2 gas concentration (1-3 mg/l), relative humidity (RH, 30-70%) and exposure time (30-90 min) were investigated using a response surface methodology (RSM). A three-factor Box-Behnken experimental design was used. The obtained data were adequately fitted to a second-order polynomial model with an R2adj of 0.992. The ClO2 gas concentration, RH and exposure time all significantly (Pgas concentration and RH as well as that between the exposure time and RH indicated significant and synergistic effects (Pgas. The inactivation of indoor biological contaminants plays an important role in preventing the transmission of pathogens and ensuring human safety. The predictive model using response surface methodology indicates the influence and interaction of the main factors on the inactivation of Bacillus subtilis subsp. niger spores by ClO2 gas, and can predict a ClO2 gas treatment condition to achieve an effective sterilization of enclosed spaces. The results in this paper will provide a reference for the application of ClO2 gas treatments for indoor disinfection.

  13. Germination and inactivation of Bacillus coagulans and Alicyclobacillus acidoterrestris spores by high hydrostatic pressure treatment in buffer and tomato sauce.

    Science.gov (United States)

    Vercammen, Anne; Vivijs, Bram; Lurquin, Ine; Michiels, Chris W

    2012-01-16

    Acidothermophilic bacteria like Alicyclobacillus acidoterrestris and Bacillus coagulans can cause spoilage of heat-processed acidic foods because they form spores with very high heat resistance and can grow at low pH. The objective of this work was to study the germination and inactivation of A. acidoterrestris and B. coagulans spores by high hydrostatic pressure (HP) treatment at temperatures up to 60°C and both at low and neutral pH. In a first experiment, spores suspended in buffers at pH 4.0, 5.0 and 7.0 were processed for 10min at different pressures (100-800MPa) at 40°C. None of these treatments caused any significant inactivation, except perhaps at 800MPa in pH 4.0 buffer where close to 1 log inactivation of B. coagulans was observed. Spore germination up to about 2 log was observed for both bacteria but occurred mainly in a low pressure window (100-300MPa) for A. acidoterrestris and only in a high pressure window (600-800MPa) for B. coagulans. In addition, low pH suppressed germination in A. acidoterrestris, but stimulated it in B. coagulans. In a second series of experiments, spores were treated in tomato sauce of pH 4.2 and 5.0 at 100 - 800MPa at 25, 40 and 60°C for 10min. At 40°C, results for B. coagulans were similar as in buffer. For A. acidoterrestris, germination levels in tomato sauce were generally higher than in buffer, and showed little difference at low and high pressure. Remarkably, the pH dependence of A. acidoterrestris spore germination was reversed in tomato sauce, with more germination at the lowest pH. Furthermore, HP treatments in the pH 4.2 sauce caused between 1 and 1.5 log inactivation of A. acidoterrestris. Germination of spores in the high pressure window was strongly temperature dependent, whereas germination of A. acidoterrestris in the low pressure window showed little temperature dependence. When HP treatment was conducted at 60°C, most of the germinated spores were also inactivated. For the pH 4.2 tomato sauce, this

  14. 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. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  15. 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.

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

    International Nuclear Information System (INIS)

    Fernandes, Joao C.; Eaton, Peter; Gomes, Ana M.; Pintado, Manuela E.; Xavier Malcata, F.

    2009-01-01

    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.

  17. Biosynthesis of silver nanoparticles by Bacillus stratosphericus spores and the role of dipicolinic acid in this process.

    Science.gov (United States)

    Hosseini-Abari, Afrouzossadat; Emtiazi, Giti; Lee, Sang-Hyuk; Kim, Byung-Gee; Kim, June-Hyung

    2014-09-01

    Seeking for simple, rapid, and environmental-friendly routes to produce metal nanoparticles is quite attractive for various biotechnological applications. Biological synthesis method of silver nanoparticles has been found very promising due to their non-toxicity and simplicity. Here, the spores of Bacillus stratosphericus isolated from soil enriched with 30 % H2O2 were used for the production of silver nanoparticles. Furthermore, the possible mechanism of silver nanoparticle synthesis by the spores was elucidated for the first time. In this regard, dipicolinic acid (DPA) was shown to play a critical role as a nanoparticle-producing agent. UV-Vis absorption spectroscopy, X-ray diffraction technique, energy-dispersive spectroscopy, and transmission electron microscopy were used to characterize the nanoparticles. Unlike vegetative cells of B. stratosphericus, the spores and the purified DPA were capable of producing nanoparticles from silver nitrate (AgNO3). These biogenic nanoparticles, which were highly toxic against different pathogenic bacteria, showed mixed structures including spherical, triangular, cubic, and hexagonal with the approximate size between 2 and 20 nm in diameter. Our results illustrated the role of dipicolinic acid as a main factor for the synthesis of nanoparticles by the bacterial spores.

  18. Radiosensibilisation of bacteria on beef minced by essential oils with special reference to the spores of Bacillus cereus ATCC 7004

    International Nuclear Information System (INIS)

    Ayari, Samia

    2007-01-01

    The radiosensitization of Bacillus Cereus ATCC 7004 spores was evaluated in the presence of thymol, thyme, D-L menthol, trans-cinnamaldehyde and eugenol in ground beef. Meat cattle minced (5 % fat) was inoculated with spores of Bacillus Cereus (10 5 - 10 6 CFU / g), and each compound was added separately at various concentrations. The antimicrobial potential was evaluated in unirradiated meat by determining the MIC in percentage (wt / wt) after 24 h of storage at 4± 1C. Results showed that the best antimicrobial compound was the trans-cinnamaldehyde with MIC of 1.47%, wt/wt. In presence of cinnamaldehyde, the addition of sodium pyrophosphate decahydrate (0.1%, wt/wt) increased significantly (p < 0.05) the relative sensitivity of Bacillus Cereus spores 2 times. However, the presence of ascorbic acid in the media reduced significantly (p < 0.05) the radiosensitivity of bacteria. The combined effect of gamma irradiation in presence of cinnamaldehyde, added with ascorbic acid or sodium pyrophosphate decahydrate, on the microbiological and physico-chemical characteristic of meat samples was evaluated at 2 kGy under air. The use of the active compounds with the irradiation reduced significantly (p < 0.05) the count of total bacteria with a concomitant effect in the extension periods of shelf life. The addition of the cinnamaldehyde induced a significant reduction (p < 0.05) in TVN and free amino acids of irradiated samples. In presence of ascorbic acid the thiobarbituric acid-reactive substances (TBARS) concentration was significantly reduced (P...0.05). A significant reduction (p < 0.05) of a* and C* of color values and a significant increase (p < 0.05 ) of b* value were obtained for the samples treated by the cinnamaldehyde. The application of bioactive films for the immobilization of the essential oils is a good alternate to check their stability during storage time. (Author). 155 refs

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

    Science.gov (United States)

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

    2016-01-01

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

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

    International Nuclear Information System (INIS)

    Zamola, B.; Karminski-Zamola, G.; Fuks, Z.; Kubovic, M.; Wrishcer, M.

    1985-01-01

    Irradiation of spore-endotoxin mixtures from Bacillus thuringiensis cultures at 254 nm (60 μW cm -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). (author)

  1. Bacillus subtilis spores PROTECT experiment Space-exposed and Mars-exposed vs. Earth-control

    Data.gov (United States)

    National Aeronautics and Space Administration — Because of their ubiquity and resistance to spacecraft decontamination bacterial spores are considered likely potential forward contaminants on robotic missions to...

  2. 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 biochemical...

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

    Science.gov (United States)

    Guidi, Valeria; Lehner, Angelika; Lüthy, Peter; Tonolla, Mauro

    2013-01-01

    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.

  4. Immune response induced by oral delivery of Bacillus subtilis spores expressing enolase of Clonorchis sinensis in grass carps (Ctenopharyngodon idellus).

    Science.gov (United States)

    Jiang, Hongye; Chen, Tingjin; Sun, Hengchang; Tang, Zeli; Yu, Jinyun; Lin, Zhipeng; Ren, Pengli; Zhou, Xinyi; Huang, Yan; Li, Xuerong; Yu, Xinbing

    2017-01-01

    Clonorchiasis, caused by the consumption of raw or undercooked freshwater fish containing infective metacercariae of Clonorchis sinensisis (C.sinensis), remains a common public health problem. New effective prevention strategies are still urgent to control this food-borne infectious disease. The previous studies suggested Bacillus subtilis (B. subtilis) spores was an ideal vaccines delivery system, and the C.sinensis enolase (CsENO) was a potential vaccine candidate against clonorchiasis. In the current study, we detected CsENO-specific IgM levels by ELISA in sera, intestinal mucus and skin mucus in grass carps (Ctenopharyngodon idella) through oral administration with B. subtilis spores surface expressing CsENO. In addition, immune-related genes expression was also measured by qRT-PCR. Grass carps orally treated with B. subtilis spores or normal forages were used as controls. The results of ELISA manifested that specific IgM levels of grass carps in CsENO group in sera, intestine mucus and skin mucus almost significantly increased from week 4 post the first oral administration when compared to the two control groups. The levels of specific IgM reached its peak in intestine mucus firstly, then in sera, and last in skin mucus. qRT-PCR results showed that 5 immune-related genes expression had different degree of rising trend in CsENO group when compared to the two control groups. Our study demonstrated that orally administrated with B. subtilis spores expressing CsENO induced innate and adaptive immunity, systemic and local mucosal immunity, and humoral and cellular immunity. Our work may pave the way to clarify the exact mechanisms of protective efficacy elicited by B. subtilis spores expressing CsENO and provide new ideas for vaccine development against C. sinensis infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. 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.

  6. 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

    subtilis mutants to probe mechanisms of spore germination and inactivation. We employ techniques of high-resolution atomic force microscopy and phase contrast microscopy to examine the effects of γ-irradiation on bacterial spores of Bacillus anthracis, Bacillus thuringiensis, and Bacillus atrophaeus spp. and of ClO2 on B. subtilis spores, and present in detail assays using spore bio-indicators to ensure sterility when decontaminating with ClO2. PMID:26322021

  7. Analysis of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores.

    NARCIS (Netherlands)

    Melis, van Clint; Nierop Groot, Masja; Tempelaars, Marcel; Moezelaar, Roy; Abee, Tjakko

    2010-01-01

    Sorbic acid (SA) is widely used as a preservative, but the effect of SA on spore germination and outgrowth has gained limited attention up to now. Therefore, the effect of sorbic acid on germination of spores of B. cereus strain ATCC 14579 was analyzed both at phenotype and transcriptome level.

  8. 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...

  9. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. 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.

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

    Science.gov (United States)

    Wendling, Morgan; Richter, William; Lastivka, Andrew; Mickelsen, Leroy

    2016-01-01

    The primary goal of this study was to determine the conditions required for the effective inactivation of Bacillus anthracis spores on materials by using methyl bromide (MeBr) gas. Another objective was to obtain comparative decontamination efficacy data with three avirulent microorganisms to assess their potential for use as surrogates for B. anthracis Ames. Decontamination tests were conducted with spores of B. anthracis Ames and Geobacillus stearothermophilus, B. anthracis NNR1Δ1, and B. anthracis Sterne inoculated onto six different materials. Experimental variables included temperature, relative humidity (RH), MeBr concentration, and contact time. MeBr was found to be an effective decontaminant under a number of conditions. This study highlights the important role that RH has when fumigation is performed with MeBr. There were no tests in which a ≥6-log10 reduction (LR) of B. anthracis Ames was achieved on all materials when fumigation was done at 45% RH. At 75% RH, an increase in the temperature, the MeBr concentration, or contact time generally improved the efficacy of fumigation with MeBr. This study provides new information for the effective use of MeBr at temperatures and RH levels lower than those that have been recommended previously. The study also provides data to assist with the selection of an avirulent surrogate for B. anthracis Ames spores when additional tests with MeBr are conducted. PMID:26801580

  12. 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.

  13. Mechanism and site of inhibition of Bacillus cereus spore outgrowth by nitrosothiols

    International Nuclear Information System (INIS)

    Morris, S.L.

    1982-01-01

    Structure vs. activity studies demonstrate that nitrosothiols inhibit outgrowth of B. cereus spores by reversible covalent bond formation with sensitive spore components. Kinetic studies of the binding of nitrosothiols and iodoacetate, a known sulfhydryl reagent, show that they complete for the same spore sites. Since two other nitrite derivatives, the Perigo factor and the transferrin inhibitor, interfere with iodoacetate label uptake in a kinetically similar fashion, all of these compounds may inhibit spore outgrowth by interacting with the same spore thiol groups. Disruption of spores which have been inhibited by radioactive iodoacetate demonstrates that much of the label is incorporated into a membrane-rich fraction that sediments as a single peak on a sucrose density gradient. SDS gel electrophoresis and autofluorography allows the identification of four intensely labelled proteins with molecular weights of 13,000, 28,000, 29,000, and 30,000. If the iodoacetate labelling is carried out in the presence of nitrosothiol, incorporation is greatly reduced into all components. When germinating spores are labelled with succinate or the lactose analog, o-nitrophenylgalactopyranoside, a significant reduction in the amount of label bound is also observed suggesting that two iodoacetate-reactive sites may be the succinate and lactose permease systems. Severe decreases in the transport of succinate and lactose into iodoacetate and nitrosothiol inhibited spores further implicates a nitrosothiol (iodoacetate) permease interaction. Iodoacetate and nitrosothiols therefore may exert their inhibitory effects by interfering with critical membrane protein sulfhydryl groups, possibly by a a covalent modification mechanism. Some of these sensitive thiols may be involved in active transport processes

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

    Science.gov (United States)

    2014-09-18

    contains an antibiotic resistance gene, which gives the cell an advantage when it keeps the plasmid. Studies will be conducted on Ba spores and...are extremely resistant to the environment and can survive in soil for decades [2]. Since the spore contains few energy compounds such as ATP and...glucose, and 10mM MgCl2. The solution was incubated for 1 hour so expression of the antibiotic resistance could occur prior to plating on selective

  15. Most of the propeptide is dispensable for stability and autoprocessing of the zymogen of the germination protease of spores of Bacillus species

    DEFF Research Database (Denmark)

    Pedersen, Lotte Bang; Nessi, C; Setlow, P

    1997-01-01

    Loss of 3, 7, or 10 of the amino-terminal 15 residues removed upon autoactivation of the zymogen of the germination protease (GPR), which initiates protein degradation during germination of spores of Bacillus species, did not result in significant changes in (i) the lack of enzymatic activity of ...

  16. Control of bacillus cereus spore germination and outgrowth in cooked rice during chilling by nonorganic and organic appled, orange, and potato peel powders

    Science.gov (United States)

    The inhibition of Bacillus cereus spore germination and outgrowth in cooked rice by nine fruit and vegetable peel powders prepared from store-bought conventional (nonorganic) and organic apples, oranges, and potatoes was investigated. The powders were mixed into rice at 10% (wt/wt) along with heat ...

  17. 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.

  18. 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 log 10 from iron and cement-mortar, respectively. Log 10 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 log 10 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 log 10 on cement-mortar. Published by Elsevier Ltd.

  19. Efficacy of Oritavancin in a Murine Model of Bacillus anthracis Spore Inhalation Anthrax

    National Research Council Canada - National Science Library

    Heine, H. S; Bassett, J; Miller, L; Bassett, A; Ivins, B. E; Lehous, D; Arhin, F. F; Parr, Jr., T. R; Moeck, G

    2008-01-01

    The inhaled form of Bacillus anthracis infection may be fatal to humans. The current standard of care for inhalational anthrax postexposure prophylaxis is ciprofloxacin therapy twice daily for 60 days...

  20. Photoproduct formation and repair capacity in a mutant of Bacillus cereus 569 producing UV-sensitive spores

    International Nuclear Information System (INIS)

    Weinberger, S.; Evenchik, Z.; Hertman, I.; Bar-Ilan Univ., Ramat-Gan

    1982-01-01

    A mutant of Bacillus cereus 569 UV sensitive in both vegetative and sporal stages was isolated by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis followed by selection on mitomycin C. The UV-sensitive mutant designated as B. cereus 2422 exhibited normal content of dipicolinic acid (DPA) and resistance to X-rays and ethyl methanesulphonate. The photoproduct type and amount, induced by a given UV dose, was similar in either cells or spores of both the mutant 2422 and the wild-type ancestor. The mutant 2422 excised cyclobutane thymine dimers only to a limited extent (20%) as compared with 80% removal in the wild type. Removal of a spore-specific photoproduct (TDHT) during germination proceeded to a similar extent in B. cereus 2422 and the wild-type parent. However, under growing conditions, an additional removal of the TDHT was observed only in the wild-type strain. Liquid holding recovery occurred in irradiated wild-type cells, but not in mutant cells. Spontaneous revertants were isolated that regained UV resistance simultaneously in both the vegetative and sporal stage. (orig./AJ)

  1. Influences of Various Peptide Linkers on the Thermotoga maritima MSB8 Nitrilase Displayed on the Spore Surface of Bacillus subtilis.

    Science.gov (United States)

    Chen, Huayou; Chen, Zhi; Wu, Bangguo; Ullah, Jawad; Zhang, Tianxi; Jia, Jinru; Wang, Hongcheng; Tan, Tianwei

    2017-01-01

    In the present study, fusion genes composed of Thermotoga maritima MSB8 nitrilase and Bacillus subtilis 168 outer coat protein CotG were constructed with various peptide linkers and displayed on B. subtilis DB 403 spores. The successful display of CotG-nit fusion proteins on the spore surface of B. subtilis was verified by Western blot analysis and activity measurement. It was demonstrated that the fusion with linker GGGGSEAAAKGGGGS presented the highest thermal and pH stability, which is 2.67- and 1.9-fold of the fusion without linker. In addition, fusion with flexible linker (GGGGS)3 demonstrated better thermal and pH stability than fusions with linkers GGGGS and (GGGGS)2. Fusion with rigid linker (EAAAK) demonstrated better thermal stability than fusions with linkers (EAAAK)2 and (EAAAK)3. Fusions with linker (EAAAK)2 demonstrated better pH stability than fusions with linkers (EAAAK) and (EAAAK)3. In the presence of 1 mM dithiothreitol, 1% (v/v) sodium dodecyl sulfate, and 20% (v/v) ethanol, the optimal linkers of the fusions were MGSSSN, GGGGSEAAAKGGGGS, and (GGGGS)3, respectively. In summary, our results showed that optimizing the peptide linkers with different type, length, and amino acid composition of the fusion proteins would be an efficient way to maintain the stability of fusion proteins and thus improve the nitrilase display efficiency, which could provide an effective method for rational design peptide linkers of displayed nitrilase on B. subtilis. © 2017 S. Karger AG, Basel.

  2. YtkD and MutT protect vegetative cells but not spores of Bacillus subtilis from oxidative stress.

    Science.gov (United States)

    Castellanos-Juárez, Francisco X; Alvarez-Alvarez, Carlos; Yasbin, Ronald E; Setlow, Barbara; Setlow, Peter; Pedraza-Reyes, Mario

    2006-03-01

    ytkD and mutT of Bacillus subtilis encode potential 8-oxo-dGTPases that can prevent the mutagenic effects of 8-oxo-dGTP. Loss of YtkD but not of MutT increased the spontaneous mutation frequency of growing cells. However, cells lacking both YtkD and MutT had a higher spontaneous mutation frequency than cells lacking YtkD. Loss of either YtkD or MutT sensitized growing cells to hydrogen peroxide (H2O2) and t-butylhydroperoxide (t-BHP), and the lack of both proteins sensitized growing cells to these agents even more. In contrast, B. subtilis spores lacking YtkD and MutT were not sensitized to H2O2, t-BHP, or heat. These results suggest (i) that YtkD and MutT play an antimutator role and protect growing cells of B. subtilis against oxidizing agents, and (ii) that neither YtkD nor MutT protects spores against potential DNA damage induced by oxidative stress or heat.

  3. 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...

  4. 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...

  5. 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.

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

    International Nuclear Information System (INIS)

    Jeong, Youngsu; Lee, Jonghee; Kim, Seongsoo

    2013-01-01

    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

  7. 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

  8. Germination inhibition of Bacillus cereus spores: impact of the lipophilic character of inhibiting compounds.

    Science.gov (United States)

    van Melis, C C J; Almeida, C Bravo; Kort, R; Groot, M N Nierop; Abee, T

    2012-11-15

    In this study, the impact of a range of organic acids and structurally similar alcohols with three to six carbon backbones and increasing lipophilic character, were tested on the germination behavior of B. cereus ATCC 14579 spores. This approach allowed substantiating whether the effectivity of the various compounds was largely dictated by membrane interference or a classic weak acid acidification effect. The octanol-water partition coefficient (log P(oct/water)) ranges from 0.25/0.33 to 2.03/1.96 for propanol/undissociated propionic acid and hexanol/undissociated hexanoic acid, respectively. Performance of germination assays at neutral (pH7) and acidic conditions (pH5.5) allowed for a comparative analysis of the action of dissociated versus undissociated acids, and the presumed pH-independent effect of the corresponding alcohols. Germination assays, based on both continuously measured optical density and time-based plating experiments, and microscopic observations demonstrated the correlation between the lipophilic character of the selected compounds and their inhibiting effect on spore germination. Real-time fluorescence based assays showed that membrane integrity in dormant spores was maintained in the presence of the tested inhibitors. Lowering the critical concentration of inhibitors by a one-step washing procedure resulted in the onset of nutrient-induced germination, indicating the reversible nature of the inhibition process. Furthermore, blocking of nutrient-induced germination in the presence of inhibitory concentrations of selected lipophilic acids and corresponding alcohols was by-passed upon addition of Ca-dipicolinic acid, pointing to loss of signaling capacity in germinant receptor-mediated germination activity. These findings show that lipophilicity is an important determinant for the ability of the selected acids and corresponding alcohols to accumulate in the spore inner membrane and their ability to act as a germination-inhibitor. Copyright

  9. 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.

  10. The Photochemistry of Unprotected DNA and DNA inside Bacillus subtilis Spores Exposed to Simulated Martian Surface Conditions of Atmospheric Composition, Temperature, Pressure, and Solar Radiation.

    Science.gov (United States)

    Nicholson, Wayne L; Schuerger, Andrew C; Douki, Thierry

    2018-03-28

    DNA is considered a potential biomarker for life-detection experiments destined for Mars. Experiments were conducted to examine the photochemistry of bacterial DNA, either unprotected or within Bacillus subtilis spores, in response to exposure to simulated martian surface conditions consisting of the following: temperature (-10°C), pressure (0.7 kPa), atmospheric composition [CO 2 (95.54%), N 2 (2.7%), Ar (1.6%), O 2 (0.13%), and H 2 O (0.03%)], and UV-visible-near IR solar radiation spectrum (200-1100 nm) calibrated to 4 W/m 2 of UVC (200-280 nm). While the majority (99.9%) of viable spores deposited in multiple layers on spacecraft-qualified aluminum coupons were inactivated within 5 min, a detectable fraction survived for up to the equivalent of ∼115 martian sols. Spore photoproduct (SP) was the major lesion detected in spore DNA, with minor amounts of cyclobutane pyrimidine dimers (CPD), in the order TT CPD > TC CPD > CT CPD. In addition, the (6-4)TC, but not the (6-4)TT, photoproduct was detected in spore DNA. When unprotected DNA was exposed to simulated martian conditions, all photoproducts were detected. Surprisingly, the (6-4)TC photoproduct was the major photoproduct, followed by SP ∼ TT CPD > TC CPD > (6-4)TT > CT CPD > CC CPD. Differences in the photochemistry of unprotected DNA and spore DNA in response to simulated martian surface conditions versus laboratory conditions are reviewed and discussed. The results have implications for the planning of future life-detection experiments that use DNA as the target, and for the long-term persistence on Mars of forward contaminants or their DNA. Key Words: Bacillus subtilis-DNA-Mars-Photochemistry-Spore-Ultraviolet. Astrobiology 18, xxx-xxx.

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

    Science.gov (United States)

    2014-06-01

    Bacillus. John Wiley and Sons, Chichester, United Kingdom. 18. Steinmetz M, Richter R. 1994. Plasmids designed to alter the antibiotic resistance ...Abigail Perez-Valdespino, Department of Biochemistry, Escuela Nacional de Ciencas Biológicas del Instituto Politécnico Nacional, Mexico City, Mexico ...contains plasmid pUB110, providing resistance to kanamycin (10 g/ml); (ii) PS3411 (4) (termed1spoVA), in which the spoVA operon is under the control of

  12. Safety assesment of Bacillus clausii UBBC07, a spore forming probiotic

    Directory of Open Access Journals (Sweden)

    Suvarna G. Lakshmi

    Full Text Available Probiotics are vital bacteria that colonize the intestine and modify its microflora with benefits for the host. Very few members of the Bacillus group are recognized as safe for use and hence only a few strains are available as commercial preparations for application in humans and animals. Acute and subacute studies in rats were conducted to establish safety of Bacillus clausii (B. clausii UBBC07. In the acute toxicity study, the oral LD50 for B. clausii UBBC07 was found to be >5000 mg/kg (630 billion cfu/kg body weight. The NOAEL for B. clausii UBBC07 was found to be 1000 (126 billion cfu mg/kg body weight/day by oral route in the subacute toxicity study. There were no significant differences between control and treated groups in any of the endpoints assessed using an OECD443 or OECD407 protocol.B. clausii UBBC07 was found to be resistant to three antibiotics −clindamycin, erythromycin and chloramphenicol. Analysis of the whole genome sequence of B. clausii UBBC07 revealed that the antibiotic resistance genes are present in chromosomal DNA which is intrinsic and not transferable. Toxin genes were also found to be absent. These results suggest consumption of B. clausii UBBC07 is safe for humans. Keywords: Acute toxicity, Subacute toxicity, NOAEL, Bacillus clausii UBBC07, Whole genome

  13. Reverse-Phase Microarray Analysis Reveals Novel Targets in Lymph Nodes of Bacillus anthracis Spore-Challenged Mice.

    Directory of Open Access Journals (Sweden)

    Taissia G Popova

    Full Text Available Anthrax is a frequently fatal infection of many animal species and men. The causative agent Bacillus anthracis propagates through the lymphatic system of the infected host; however, the specific interactions of the host and microbe within the lymphatics are incompletely understood. We report the first description of the phosphoprotein signaling in the lymph nodes of DBA/2 mice using a novel technique combining the reverse-phase microarray with the laser capture microdissection. Mice were challenged into foot pads with spores of toxinogenic, unencapsulated Sterne strain. The spores quickly migrated to the regional popliteal lymph nodes and spread to the bloodstream as early as 3 h post challenge. All mice died before 72 h post challenge from the systemic disease accompanied by a widespread LN tissue damage by bacteria, including the hemorrhagic necrotizing lymphadenitis, infiltration of CD11b+ and CD3+ cells, and massive proliferation of bacteria in lymph nodes. A macrophage scavenger receptor CD68/macrosialin was upregulated and found in association with vegetative bacteria likely as a marker of their prior interaction with macrophages. The major signaling findings among the 65 tested proteins included the reduced MAPK signaling, upregulation of STAT transcriptional factors, and altered abundance of a number of pro- and anti-apoptotic proteins with signaling properties opposing each other. Downregulation of ERK1/2 was associated with the response of CD11b+ macrophages/dendritic cells, while upregulation of the pro-apoptotic Puma indicated a targeting of CD3+ T-cells. A robust upregulation of the anti-apoptotic survivin was unexpected because generally it is not observed in adult tissues. Taken together with the activation of STATs it may reflect a new pathogenic mechanism aimed to delay the onset of apoptosis. Our data emphasize a notion that the net biological outcome of disease is determined by a cumulative impact of factors representing the

  14. Reverse-Phase Microarray Analysis Reveals Novel Targets in Lymph Nodes of Bacillus anthracis Spore-Challenged Mice.

    Science.gov (United States)

    Popova, Taissia G; Espina, Virginia; Liotta, Lance A; Popov, Serguei G

    2015-01-01

    Anthrax is a frequently fatal infection of many animal species and men. The causative agent Bacillus anthracis propagates through the lymphatic system of the infected host; however, the specific interactions of the host and microbe within the lymphatics are incompletely understood. We report the first description of the phosphoprotein signaling in the lymph nodes of DBA/2 mice using a novel technique combining the reverse-phase microarray with the laser capture microdissection. Mice were challenged into foot pads with spores of toxinogenic, unencapsulated Sterne strain. The spores quickly migrated to the regional popliteal lymph nodes and spread to the bloodstream as early as 3 h post challenge. All mice died before 72 h post challenge from the systemic disease accompanied by a widespread LN tissue damage by bacteria, including the hemorrhagic necrotizing lymphadenitis, infiltration of CD11b+ and CD3+ cells, and massive proliferation of bacteria in lymph nodes. A macrophage scavenger receptor CD68/macrosialin was upregulated and found in association with vegetative bacteria likely as a marker of their prior interaction with macrophages. The major signaling findings among the 65 tested proteins included the reduced MAPK signaling, upregulation of STAT transcriptional factors, and altered abundance of a number of pro- and anti-apoptotic proteins with signaling properties opposing each other. Downregulation of ERK1/2 was associated with the response of CD11b+ macrophages/dendritic cells, while upregulation of the pro-apoptotic Puma indicated a targeting of CD3+ T-cells. A robust upregulation of the anti-apoptotic survivin was unexpected because generally it is not observed in adult tissues. Taken together with the activation of STATs it may reflect a new pathogenic mechanism aimed to delay the onset of apoptosis. Our data emphasize a notion that the net biological outcome of disease is determined by a cumulative impact of factors representing the microbial insult and

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

    Science.gov (United States)

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

    2017-07-01

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

  16. The recombinant fusion protein of cholera toxin B and neutrophil-activating protein expressed on Bacillus subtilis spore surface suppresses allergic inflammation in mice.

    Science.gov (United States)

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

    2017-07-01

    The neutrophil-activating protein of Helicobacter pylori (HP-NAP) has been identified as a modulator with anti-Th2 inflammation activity, and cholera toxin B (CTB) is a mucosal adjuvant that can also induce antigen tolerance. In this study, we constructed a CTB-NAP fusion protein on the surface of Bacillus subtilis spore and evaluate the efficiency of oral administration of the recombinant CTB-NAP spores in preventing asthma in mice. Oral administration of recombinant CTB or CTB-NAP spores significantly decreased serum ovalbumin (OVA)-specific IgE (p recombinant spores. Oral administration of recombinant CTB or CTB-NAP spores induced IL-10 and IFN-γ expression and reduced IL-4 levels in bronchoalveolar lavage fluid (BALF). Moreover, CTB and CTB-NAP spores reduced the eosinophils in BALF and inflammatory cell infiltration in the lungs. Furthermore, CD4 + CD25 + Foxp3 + Tregs in splenocytes were significantly increased in mice treated with recombinant CTB or CTB-NAP spores. The number of CD4 + CD25 + Foxp3 + Tregs caused by CTB-NAP was higher than that by CTB alone. Our study indicated that B. subtilis spores with surface expression of subunit CTB or CTB-NAP could inhibit OVA-induced allergic inflammation in mice. The attenuated inflammation was attributed to the induction of CD4 + CD25 + Foxp3 + Tregs and IgA. Moreover, the fusion protein CTB-NAP demonstrated a better efficiency than CTB alone in inhibiting the inflammation.

  17. 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.

  18. Effect of coexisting organic substances on radiation resistance of Bacillus pumilus spores suspended in water

    International Nuclear Information System (INIS)

    Kigawa, Akiko; Tateishi, Tsuneo; Iso, Katsuaki; Kimura, Toshio; Mamuro, Tetsuo

    1987-01-01

    D values of B. pumilus spores suspended in water have been shown to increase in the presence of some coexisting organic substances. For elucidation of a mechanism or mechanisms involved in such a phenomenon, D-values of B.p. spores were examined by suspending them in aqueous solutions containing various concentrations of ethanol, glycerin, inulin and PVA. All these substances showed abrupt changes in D value at a narrow concentration range of 1 - 10 weight ppm. Solutions containing these substances at their lower limit concentrations and upper limit were prepared, sealed in incubator bottles leaving no air layer and irradiated at 0.7 Mrad with γ-rays. Winkler's method was used for the determination of oxygen concentrations in these solutions. The initial concentration of dissolved oxygen was 8.2 ppm. After irradiation, 3 - 5 ppm of oxygen remained in those solutions containing the lower limit (1 ppm), whereas only less than 0.5 ppm in those containing the upper limits, 2.5 ppm of ethanol, 5 ppm of PVA and 10 ppm each of glycerin and inulin. Therefore, the observed effect of coexisting organic substances on radiation resistance of B. pumilus can be explained by the so-called ''oxygen effect''. (author)

  19. Prevalence of Vibrio cholerae in Coastal Alternative Supplies of Drinking Water and Association with Bacillus-Like Spore Formers

    Directory of Open Access Journals (Sweden)

    Md. Asaduzzaman Shishir

    2018-02-01

    Full Text Available The scarcity of hygienic drinking water is a normal phenomenon in the coastal areas of Bangladesh due to the high salinity of ground water. The inhabitants of this locality, therefore, live on alternative supplies of water including rain-fed pond water, and rainwater with persistent complex microbial interactions therein, often contaminated with life-threatening pathogens. Hence, this study was aimed at analyzing the prevalence of Vibrio cholerae (Vc in the alternative drinking waters of Mathbaria, a coastal subdistrict neighboring the Bay of Bengal, the efficacy of pond sand filter (PSF and the co-association among Bacillus-like spore formers (Sf and Vc. Vc presumably entrapped into the membrane filter was enriched in alkaline peptone water medium and was isolated on selective thiosulfate-citrate-bile salts-sucrose and taurocholate-tellurite-gelatin agar media. They were finally identified by immunochromatographic one step rapid test and serology test. A total of 26% Vc positive samples were obtained out of 100 [ponds—48, household (HH—29, and PSFs—23] where 13% cases were pathogenic (Vc O1 and 13% were non-pathogenic (Vc non-O1/non-O139. The distribution of Vc as observed was 33, 26, and 13.8% in waters derived from pond surface, PSF, and HH reservoirs, respectively, and for pathogenic type, it was 62.5%, 50%, and nil, respectively. Although none of the samples was identified with pathogenic Vc O139, the statistics represents a significant and augmentative risk of cholera outbreak in the focused area. The antibiotic sensitivity pattern in this study resembled the trend observed during last few years for Vc. The PSF demonstrated its inability to remove Vc from any of the samples and in addition, the filter itself was evidenced to be the source of pathogens and spores in further contamination and transmission. The development of biofilm in the PSF could be hypothesized as the reservoir in contaminating pathogen-free water samples. From the

  20. 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; Pillai, Segaran P

    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 10(6) spores/mL (ca. 1.5 × 10(5) 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.

  1. Role Played by Exosporium Glycoproteins in the Surface Properties of Bacillus cereus Spores and in Their Adhesion to Stainless Steel ▿

    Science.gov (United States)

    Lequette, Yannick; Garénaux, Estelle; Tauveron, Grégoire; Dumez, Sylvain; Perchat, Stéphane; Slomianny, Christian; Lereclus, Didier; Guérardel, Yann; Faille, Christine

    2011-01-01

    Bacillus cereus spores are surrounded by a loose-fitting layer called the exosporium, whose distal part is mainly formed from glycoproteins. The role played by the exosporium glycoproteins of B. cereus ATCC 14579 (BclA and ExsH) was investigated by considering hydrophobicity and charge, as well as the properties of spore adhesion to stainless steel. The absence of BclA increased both the isoelectric point (IEP) and hydrophobicity of whole spores while simultaneously reducing the interaction between spores and stainless steel. However, neither the hydrophobicity nor the charge associated with BclA could explain the differences in the adhesion properties. Conversely, ExsH, another exosporium glycoprotein, did not play a significant role in spore surface properties. The monosaccharide analysis of B. cereus ATCC 14579 showed different glycosylation patterns on ExsH and BclA. Moreover, two specific glycosyl residues, namely, 2-O-methyl-rhamnose (2-Me-Rha) and 2,4-O-methyl-rhamnose (2,4-Me-Rha), were attached to BclA, in addition to the glycosyl residues already reported in B. anthracis. PMID:21622795

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

    Science.gov (United States)

    Pandey, Rachna; Pieper, Gerard H; Ter Beek, Alexander; Vischer, Norbert O E; Smelt, Jan P P M; Manders, Erik M M; Brul, Stanley

    2015-12-01

    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 the food industry. Its effect on spore germination and outgrowth in a combined, 'hurdle', preservation setting has gained limited attention. Therefore, the effects of mild sorbic acid (3 mM), heat-treatment (85 °C for 10 min) and a combination of both mild stresses on germination and outgrowth of B. subtilis 1A700 spores were analysed at single spore level. The heat-treatment of the spore population resulted in a germination efficiency of 46.8% and an outgrowth efficiency of 32.9%. In the presence of sorbic acid (3 mM), the germination and outgrowth efficiency was 93.3% and 80.4% respectively whereas the combined heat and sorbic acid stress led to germination and outgrowth efficiencies of 52.7% and 27.0% respectively. The heat treatment clearly primarily affected the germination process, while sorbic acid affected the outgrowth and generation time. In addition a new 'burst' time-point was defined as the time-point at which the spore coat visibly breaks and/or is shed. The combined stresses had a synergistic effect on the time of the end of germination to the burst time-point, increasing both the mean and its variation more than either of the single stresses did. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Diversity of spore-forming bacteria and identification of Bacillus amyloliquefaciens as a species frequently associated with the ropy spoilage of bread.

    Science.gov (United States)

    Valerio, F; De Bellis, P; Di Biase, M; Lonigro, S L; Giussani, B; Visconti, A; Lavermicocca, P; Sisto, A

    2012-06-01

    This study examines the diversity of spore-forming bacteria isolated from raw materials/bread using molecular methods along with a rapid and innovative technology, the FT-NIR spectroscopy. Microbiological analysis showed that 23% of semolina and 42% of other raw materials (including grain, brewer yeast, improvers) contained more than 100 spores/g and more than 50% of each kind of sample was contaminated at a level ranging from 1 to 100 spores/g. A high bacterial diversity characterized raw materials. In total 176 isolates were collected and characterized: 13 bacterial species belonging to Bacillus (10) and Paenibacillus (3) genera were identified by sequencing of 16S rRNA, gyrA or gyrB genes. The two closely related species Bacillus amyloliquefaciens (strain N45.1) and Bacillus subtilis (strain S63) were also analyzed by the spectroscopic technique FT-NIR. This analysis gave clear discrimination between the strains in the score plot obtained by the PCA and allowed to identify the spectral region 5600-4000 cm(-1) as the information-rich region for discrimination. B. amyloliquefaciens, possibly misidentified as B. subtilis in previous studies, was recognized as the most frequent species, found also in ropy bread. Moreover, the screening test for rope production indicated that mainly B. amyloliquefaciens, together with B. subtilis and Bacillus pumilus, could cause spoilage in bread, even if the last two species were represented by a low number of isolates. The Bacillus cereus group and Bacillus megaterium showed a lower percentage (30-70%) of isolates potentially able to cause the rope, but considering the high number of B. cereus group isolates detected in this study, this bacterial group should also be considered important in rope spoilage. In conclusion, results demonstrate that raw materials used to produce bread represent a rich source of spore-forming bacteria, therefore their microbiological quality should be monitored before use. Moreover, this study

  4. Detection of Bacillus anthracis spores and a model protein using PEMC sensors in a flow cell at 1 mL/min.

    Science.gov (United States)

    Campbell, Gossett A; Mutharasan, Raj

    2006-07-15

    Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors of 4mm(2) sensing area were immobilized with antibody specific to Bacillus anthracis (anti-BA) spores or bovine serum albumin (anti-BSA). Detection of pathogen (Bacillus anthracis (BA) at 300 spores/mL) and BSA (1 mg/mL) were investigated under both stagnant and flow conditions. Two flow cell designs were evaluated by characterizing flow-induced resonant frequency shifts. One of the flow cells labeled SFC-2 (hold-up volume of 0.3 mL), showed small fluctuations (+/-20 Hz) around a common resonant frequency response of 217 Hz in the flow rate range of 1-17 mL/min. The total resonant frequency change obtained for the binding of 300 spores/mL in 1h was 90+/-5 Hz (n=2), and 162+/-10 Hz (n=2) under stagnant and flow conditions, respectively. Binding of antibodies, anti-BA and anti-BSA, were more rapid under flow than under stagnant conditions. The sensor was repeatedly exposed to BSA with an intermediate release step. The first and second responses to BSA were nearly identical. The total resonant frequency response to BSA was 388+/-10 (n=2) Hz under flow conditions. Kinetic analysis is carried out to quantify the effect of flow rate on antibody immobilization and the two types of detection experiments.

  5. Response surface methodology as a tool for modeling and optimization of Bacillus subtilis spores inactivation by UV/ nano-Fe0process for safe water production.

    Science.gov (United States)

    Yousefzadeh, Samira; Matin, Atiyeh Rajabi; Ahmadi, Ehsan; Sabeti, Zahra; Alimohammadi, Mahmood; Aslani, Hassan; Nabizadeh, Ramin

    2018-04-01

    One of the most important aspects of environmental issues is the demand for clean and safe water. Meanwhile, disinfection process is one of the most important steps in safe water production. The present study aims at estimating the performance of UV, nano Zero-Valent Iron particles (nZVI, nano-Fe 0 ), and UV treatment with the addition of nZVI (combined process) for Bacillus subtilis spores inactivation. Effects of different factors on inactivation including contact time, initial nZVI concentration, UV irradiance and various aerations conditions were investigated. Response surface methodology, based on a five-level, two variable central composite design, was used to optimize target microorganism reduction and the experimental parameters. The results indicated that the disinfection time had the greatest positive impact on disinfection ability among the different selected independent variables. According to the results, it can be concluded that microbial reduction by UV alone was more effective than nZVI while the combined UV/nZVI process demonstrated the maximum log reduction. The optimum reduction of about 4 logs was observed at 491 mg/L of nZVI and 60 min of contact time when spores were exposed to UV radiation under deaerated condition. Therefore, UV/nZVI process can be suggested as a reliable method for Bacillus subtilis spores inactivation. Copyright © 2018. Published by Elsevier Ltd.

  6. Alkaliphilic Bacillus species show potential application in concrete crack repair by virtue of rapid spore production and germination then extracellular calcite formation.

    Science.gov (United States)

    Sharma, T K; Alazhari, M; Heath, A; Paine, K; Cooper, R M

    2017-05-01

    Characterization of alkaliphilic Bacillus species for spore production and germination and calcite formation as a prelude to investigate their potential in microcrack remediation in concrete. Conditions, extent and timing of endospore production was determined by dark-field light microscopy; germination induction and kinetics were assessed by combining reduction in optical density with formation of refractile bodies by phase-contrast microscopy. Bacillus pseudofirmus was selected from several species as the most suitable isolate. Levels and timing of calcium carbonate precipitated in vitro by B. pseudofirmus were evaluated by atomic absorption spectroscopy and structural identity confirmed as calcite and aragonite by Raman spectroscopy and FTIR. The isolate produced copious spores that germinated rapidly in the presence of germinants l-alanine, inosine and NaCl. Bacterial cells produced CaCO 3 crystals in microcracks and the resulting occlusion markedly restricted water ingress. By virtue of rapid spore production and germination, calcium carbonate formation in vitro and in situ, leading to sealing of microcracks, B. pseudofirmus shows clear potential for remediation of concrete on a commercial scale. Microbial sealing of microcracks should become a practicable and sustainable means of increasing concrete durability. © 2017 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of The Society for Applied Microbiology.

  7. 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. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2017-10-01

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

  9. Influência do meio suporte na inativação de endósporos de Bacillus atrophaeus em resíduos de serviços de saúde por micro-ondas

    Directory of Open Access Journals (Sweden)

    Elisa Augusta de Oliveira

    2015-06-01

    Full Text Available A Norma Técnica CETESB P2.111/2007 foi tomada como base para avaliar a desinfecção de resíduos de serviços de saúde (RSS por micro-ondas, através da comparação dos resultados provenientes da utilização da norma, ou seja, com endósporos de Bacillus atropheus em suspensão no interior de ampolas introduzidas nos RSS, com os obtidos nos endósporos dispersos na superfície das partículas. Os resultados foram ajustados à cinética de primeira ordem e à Lei de Arrhenius para obtenção das energias de ativação (Ea e dos fatores pré-exponenciais (k0. A Ea obtida nos endósporos em suspensão nas ampolas (99-45 J.mol-1 foram sensivelmente menores quando comparadas com as Ea (9.203-5.782 J.mol-1 provenientes da inativação dos endósporos dispersos sobre os RSS, para k0 praticamente constantes (0,26 e 0,23 min-1, respectivamente. Portanto, a resistência à inativação dos endósporos foi bastante afetada pelo meio suporte utilizado, tendo sido mais sensíveis à exposição a micro-ondas no meio líquido do que no meio sólido.

  10. COMPARISON OF UV INACTIVATION OF SPORES OF THREE ENCEPHALITOZOON SPECIES WITH THAT OF SPORES OF TWO DNA REPAIR-DEFICIENT BACILLUS SUBTILIS BIODOSIMETRY STRAINS

    Science.gov (United States)

    The sensitivity of three Encephalitozoon spp. to ultraviolet (UV) inactivation was determined. Encephalitozoon intestinalis is a contaminant listed on the USEPA's 1998 Contaminant Candidate List (CCL). Also, use of DNA repair deficient strains of Bacillus subtilis were evaluat...

  11. Influence of Cooling Rate on Growth of Bacillus cereus from Spore Inocula in Cooked Rice, Beans, Pasta, and Combination Products Containing Meat or Poultry.

    Science.gov (United States)

    Juneja, Vijay K; Mohr, Tim B; Silverman, Meryl; Snyder, O Peter

    2018-02-23

    The objective of this study was to assess the ability of Bacillus cereus spores to germinate and grow in order to determine a safe cooling rate for cooked rice, beans, and pasta, rice-chicken (4:1), rice-chicken-vegetables (3:1:1), rice-beef (4:1), and rice-beef-vegetables (3:1:1). Samples were inoculated with a cocktail of four strains of heat-shocked (80°C for 10 min) B. cereus spores (NCTC 11143, 935A/74, Brad 1, and Mac 1) to obtain a final spore concentration of approximately 2 log CFU/g. Thereafter, samples were exponentially cooled through the temperature range of 54.5 to 7.2°C in 6, 9, 12, 15, 18, and 21 h. At the end of the cooling period, samples were removed and plated on mannitol egg yolk polymyxin agar. The plates were incubated at 30°C for 24 h. The net B. cereus growth from spores in beans was beans, pasta, rice-chicken, rice-chicken-vegetables, rice-beef, and rice-beef-vegetables to guard against the hazards associated with B. cereus.

  12. Evaluating the Sporicidal Activity of Disinfectants againstClostridium difficileandBacillus amyloliquefaciensSpores by Using the Improved Methods Based on ASTM E2197-11.

    Science.gov (United States)

    Uwamahoro, Marie Christine; Massicotte, Richard; Hurtubise, Yves; Gagné-Bourque, François; Mafu, Akier Assanta; Yahia, L'Hocine

    2018-01-01

    Spore-forming pathogenic bacteria, such as Clostridium difficile , are associated with nosocomial infection, leading to the increased use of sporicidal disinfectants, which impacts socioeconomic costs. However, C. difficile can be prevented using microorganisms such as Bacillus amyloliquefaciens , a prophylactic agent that has been proven to be effective against it in recent tests or it can be controlled by sporicidal disinfectants. These disinfectants against spores should be evaluated according to a known and recommended standard. Unfortunately, some newly manufactured disinfectants like Bioxy products have not yet been tested. ASTM E2197-11 is a standard test that uses stainless steel disks (1 cm in diameter) as carriers, and the performance of the test formulation is calculated by comparing the number of viable test organisms to that on the control carriers. Surface tests are preferable for evaluating disinfectants with sporicidal effects on hard surfaces. This study applies improved methods, based on the ASTM E2197-11 standard, for evaluating and comparing the sporicidal efficacies of several disinfectants against spores of C. difficile and B. amyloliquefaciens , which are used as the test organisms. With the improved method, all spores were recovered through vortexing and membrane filtration. The results show that chlorine-based products are effective in 5 min and Bioxy products at 5% w/v are effective in 10 min. Although Bioxy products may take longer to prove their effectiveness, their non-harmful effects to hospital surfaces and people have been well established in the literature.

  13. 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....

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

    Science.gov (United States)

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

    2017-04-01

    Spore heat resistance, germination, and outgrowth are problematic bacterial properties compromising food safety and quality. Large interstrain variation in these properties makes prediction and control of spore behavior challenging. High-level heat resistance and slow germination of spores of some natural Bacillus subtilis isolates, encountered in foods, have been attributed to the occurrence of the spoVA 2mob operon carried on the Tn 1546 transposon. In this study, we further investigate the correlation between the presence of this operon in high-level-heat-resistant spores and their germination efficiencies before and after exposure to various sublethal heat treatments (heat activation, or HA), which are known to significantly improve spore responses to nutrient germinants. We show that high-level-heat-resistant spores harboring spoVA 2mob required higher HA temperatures for efficient germination than spores lacking spoVA 2mob The optimal spore HA requirements additionally depended on the nutrients used to trigger germination, l-alanine (l-Ala), or a mixture of l-asparagine, d-glucose, d-fructose, and K + (AGFK). The distinct HA requirements of these two spore germination pathways are likely related to differences in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved in this germination response. In contrast, no relation was found between transcription levels of main germination genes and spore germination phenotypes. The findings presented in this study have great implications for practices in the food industry, where heat treatments are commonly used to inactivate pathogenic and spoilage microbes, including bacterial spore formers. IMPORTANCE This study describes a strong variation in spore germination capacities and requirements for a heat activation treatment, i.e., an exposure to sublethal heat that increases

  15. Kinetics of Inactivation of Bacillus subtilis subsp. niger Spores and Staphylococcus albus on Paper by Chlorine Dioxide Gas in an Enclosed Space.

    Science.gov (United States)

    Wang, Tao; Wu, Jinhui; Qi, Jiancheng; Hao, Limei; Yi, Ying; Zhang, Zongxing

    2016-05-15

    Bacillus subtilis subsp. niger spore and Staphylococcus albus are typical biological indicators for the inactivation of airborne pathogens. The present study characterized and compared the behaviors of B. subtilis subsp. niger spores and S. albus in regard to inactivation by chlorine dioxide (ClO2) gas under different gas concentrations and relative humidity (RH) conditions. The inactivation kinetics under different ClO2 gas concentrations (1 to 5 mg/liter) were determined by first-order and Weibull models. A new model (the Weibull-H model) was established to reveal the inactivation tendency and kinetics for ClO2 gas under different RH conditions (30 to 90%). The results showed that both the gas concentration and RH were significantly (P 70%). Compared with the first-order model, the Weibull and Weibull-H models demonstrated a better fit for the experimental data, indicating nonlinear inactivation behaviors of the vegetative bacteria and spores following exposure to ClO2 gas. The times to achieve a six-log reduction of B. subtilis subsp. niger spore and S. albus were calculated based on the established models. Clarifying the kinetics of inactivation of B. subtilis subsp. niger spores and S. albus by ClO2 gas will allow the development of ClO2 gas treatments that provide an effective disinfection method. Chlorine dioxide (ClO2) gas is a novel and effective fumigation agent with strong oxidization ability and a broad biocidal spectrum. The antimicrobial efficacy of ClO2 gas has been evaluated in many previous studies. However, there are presently no published models that can be used to describe the kinetics of inactivation of airborne pathogens by ClO2 gas under different gas concentrations and RH conditions. The first-order and Weibull (Weibull-H) models established in this study can characterize and compare the behaviors of Bacillus subtilis subsp. niger spores and Staphylococcus albus in regard to inactivation by ClO2 gas, determine the kinetics of inactivation of

  16. High-Level Heat Resistance of Spores of Bacillus amyloliquefaciens and Bacillus licheniformis Results from the Presence of a spoVA Operon in a Tn1546 Transposon

    NARCIS (Netherlands)

    Berendsen, Erwin M; Koning, Rosella A; Boekhorst, Jos; de Jong, Anne; Kuipers, Oscar P; Wells-Bennik, Marjon H J

    2016-01-01

    Bacterial endospore formers can produce spores that are resistant to many food processing conditions, including heat. Some spores may survive heating processes aimed at production of commercially sterile foods. Recently, it was shown that a spoVA operon, designated spoVA(2mob), present on a Tn1546

  17. Recovery of heat treated Bacillus cereus spores is affected by matrix composition and factors with putative functions in damage repair

    NARCIS (Netherlands)

    Warda, A.K.; Tempelaars, M.H.; Abee, T.; Nierop Groot, M.N.

    2016-01-01

    The ability of spores to recover and grow out after food processing is affected by cellular factors and by the outgrowth conditions. In the current communication we studied the recovery and outgrowth of individually sorted spores in BHI and rice broth media and on agar plates using flow cytometry.

  18. Forces and Kinetics of the Bacillus subtilis Spore Coat Proteins CotY and CotX Binding to CotE Inspected by Single Molecule Force Spectroscopy.

    Science.gov (United States)

    Liu, Huiqing; Krajcikova, Daniela; Wang, Nan; Zhang, Zhe; Wang, Hongda; Barak, Imrich; Tang, Jilin

    2016-02-18

    Spores are uniquely stable cell types that are produced when bacteria encounter nutrient limitations. Spores are encased in a complex multilayered coat, which provides protection against environmental insults. The spore coat of Bacillus subtilis is composed of around 70 individual proteins that are organized into four distinct layers. Here we explored how morphogenetic protein CotE guides formation of the outermost layer of the coat, the crust, around the forespore by focusing on three proteins: CotE, CotY, and CotX. Single molecule force spectroscopy (SMFS) was used to investigate the interactions among CotE, CotY, and CotX at the single-molecule level. Direct interactions among these three proteins were observed. Additionally, the dissociation kinetics was also studied by measuring the unbinding forces of the complexes at different loading rates. A series of kinetic data of these complexes were acquired. It was found that the interaction of CotE and CotY was stronger than that of CotE and CotX.

  19. Comparison of the disinfection effects of vacuum-UV (VUV) and UV light on Bacillus subtilis spores in aqueous suspensions at 172, 222 and 254 nm.

    Science.gov (United States)

    Wang, Ding; Oppenländer, Thomas; El-Din, Mohamed Gamal; Bolton, James R

    2010-01-01

    The efficacy of UV and vacuum-UV (VUV) disinfection of Bacillus subtilis spores in aqueous suspensions at wavelengths of 172, 222 and 254 nm was evaluated. A Xe(2)* excilamp, a KrCl* excilamp and a low-pressure mercury lamp were used as almost monochromatic light sources at these three wavelengths. The first-order inactivation rate constants at 172, 222 and 254 nm were 0.0023, 0.122 and 0.069 cm(2) mJ(-1), respectively. Therefore, a 2 log reduction of B. subtilis spores was reached with fluences (UV doses) of 870, 21.6 and 40.4 mJ cm(-2) at these individual wavelengths. Consequently, for the inactivation of B. subtilis spores, VUV exposure at 172 nm is much less efficient than exposure at the other two wavelengths, while exposure at 222 nm is more efficient than that at 254 nm, which is probably because triplet energy transfer from DPA to thymine bases at 222 nm is higher than that at 254 nm. This research indicated quantitatively that VUV light is not practicable for microorganism disinfection in water and wastewater treatment. However, in comparison with other advanced oxidation processes (e.g. UV/TiO(2), UV/H(2)O(2) or O(3)/H(2)O(2)) the VUV-initiated photolysis of water is likely more efficient in generating hydroxyl radicals and more effective for the inactivation of microorganisms.

  20. Production of d-Allulose with d-Psicose 3-Epimerase Expressed and Displayed on the Surface of Bacillus subtilis Spores.

    Science.gov (United States)

    He, Weiwei; Jiang, Bo; Mu, Wanmeng; Zhang, Tao

    2016-09-28

    The production of d-allulose is usually conducted via isolated-enzyme or whole-cell biocatalysis reactions. In the present study, an innovative biocatalyst, d-psicose 3-epimerase (DPEase) from Clostridium scindens ATCC 35704, presented on the surface of Bacillus subtilis spores, was applied for d-allulose production. DPEase was fused at the C-terminus of the anchoring protein, CotZ, via a peptide linker, and trophic genes were used as selection markers during the chromosomal integration. The optimal temperature and pH of the fusion protein CotZ-DPEase were 55 °C and pH 7.5-8.0, respectively, and the anchored DPEase exhibited high thermostability. Under optimal conditions, 30 g/L of recombinant spores can produce 85 g/L d-allulose from 500 g/L d-fructose after 12 h, and 60% of the yield was maintained after five cycles of utilization. Therefore, this biocatalyst system, capable of expressing and immobilizing DPEase on the spore surface of B. subtilis, was an appropriate alternative for d-allulose production.

  1. Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture.

    Science.gov (United States)

    Hutchison, J R; Piepel, G F; Amidan, B G; Hess, B M; Sydor, M A; Deatherage Kaiser, B L

    2018-01-21

    We evaluated the effects of Bacillus anthracis surrogates, low surface concentrations, surface materials and assay methods on false-negative rate (FNR) and limit of detection (LOD 95 ) for recovering Bacillus spores using a macrofoam-swab sampling procedure. Bacillus anthracis Sterne or Bacillus atrophaeus Nakamura spores were deposited over a range of low target concentrations (2-500 per coupon) onto glass, stainless steel, vinyl tile and plastic. Samples were assayed using a modified Rapid Viability-PCR (mRV-PCR) method and the traditional plate culture method to obtain FNR and LOD 95 results. Mean FNRs tended to be lower for mRV-PCR compared to culturing, and increased as spore concentration decreased for all surface materials. Surface material, but not B. anthracis surrogate, influenced FNRs with the mRV-PCR method. The mRV-PCR LOD 95 was lowest for glass and highest for vinyl tile. LOD 95 values overall were lower for mRV-PCR than for the culture method. This study adds to the limited data on FNR and LOD 95 for mRV-PCR and culturing methods with low concentrations of B. anthracis sampled from various surface materials by the CDC macrofoam-swab method. These are key inputs for planning characterization and clearance studies for low contamination levels of B. anthracis. © 2018 The Society for Applied Microbiology.

  2. Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture

    Energy Technology Data Exchange (ETDEWEB)

    Hutchison, Janine R.; Piepel, Gregory F.; Amidan, Brett G.; Hess, Becky M.; Sydor, Michael A.; Kaiser, Brooke LD

    2018-03-13

    Aims: We evaluated the effects of Bacillus anthracis surrogates, low surface concentrations, surface materials, and assay methods on false-negative rate (FNR) and limit of detection (LOD95) for recovering Bacillus spores using a macrofoam-swab sampling procedure. Methods and Results: Bacillus anthracis Sterne or Bacillus atrophaeus Nakamura spores were deposited over a range of low target concentrations (2 – 500 coupon-1) onto glass, stainless steel, vinyl tile, and plastic. Samples were assayed using a modified Rapid Viability-PCR (mRV-PCR) method and the traditional plate culture method to obtain FNR and LOD95 results. Conclusions: Mean FNRs tended to be lower for mRV-PCR compared to culturing, and increased as spore concentration decreased for all surface materials. Surface material, but not B. anthracis surrogate, influenced FNRs with the mRV-PCR method. The mRV-PCR LOD95 was lowest for glass and highest for vinyl tile. LOD95 values overall were lower for mRV-PCR than for the culture method. Significance and Impact of Study: This study adds to the limited data on FNR and LOD95 for mRV-PCR and culturing methods with low concentrations of B. anthracis sampled from various surface materials by the CDC macrofoam-swab method. These are key inputs for planning characterization and clearance studies for low contamination levels of B. anthracis.

  3. 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.

  4. 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...

  5. Antimicrobial Effects of Gold/Copper Sulphide (Au/Cus) Core/Shell Nanoparticles on Bacillus Anthracis Spores and Cells

    Science.gov (United States)

    2013-01-01

    and DNA extrusion experiments revealed that nanoparticles damaged the cell membrane causing DNA and cytosolic content efflux and eventually cell...significant spore (x 105) killing after 24 h of pre-treatment. SEM imaging, EDS analysis, and DNA extrusion experiments revealed that nanoparticles...CO2. The spores have a highly ordered structure with a multilayered proteinaceous shell called the coat. The coat is responsible for resistance and

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

    Science.gov (United States)

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

    2017-10-20

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

  7. New insights into the effectiveness of alpha-amylase enzyme presentation on the Bacillus subtilis spore surface by adsorption and covalent immobilization.

    Science.gov (United States)

    Gashtasbi, Fatemeh; Ahmadian, Gholamreza; Noghabi, Kambiz Akbari

    2014-10-01

    Most of the studies in the field of enzyme immobilization have sought to develop a simple, efficient and cost-effective immobilization system. In this study, probiotic Bacillus spores were used as a matrix for enzyme immobilization, because of their inherent resistance to extreme temperatures, UV irradiation, solvents and drying. Above all, their preparation is cost-effective. The alpha-amylase enzyme was immobilized on the spore surface by the covalent and adsorption methods. For the covalent method, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N hydroxysulfosuccinimide (NHS) were used. The maximum concentration of the alpha-amylase immobilized by the two methods onto the spore surface was 360 μg/1.2×10(11) spore. However, maximum activity was achieved at an enzyme concentration of approximately 60 μg/.4×10(10), corresponding to an estimated activity of 8×10(3) IU mg(-1)/1.2×10(11) spore for covalent immobilization and 8.53×10(3) for the adsorption method. After washing the enzyme with 1M NaCl and 0.5% Triton X-100, the enzyme immobilization yield was estimated to be 77% and 20.07% for the covalent and adsorption methods, respectively. The alpha-amylase immobilized by both methods, displayed improved activity in the basic pH range. The optimum pH for the free enzyme was 5 while it shifted to 8 for the immobilized enzyme. The optimum temperatures for the free and immobilized enzymes were 60 °C and 80 °C, respectively. The covalently-immobilized alpha-amylase retained 65% of its initial activity, even after 10 times of recycling. The Km and Vmax values were determined by the GraphPad Prism software, which showed that the Vmax value decreased moderately after immobilization. This is the first study which reports the covalent immobilization of an enzyme on the spore surface. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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. - Highlights: • Bacillus spores and MS2

  9. Experimental Design for a Macrofoam-Swab Study Relating the Recovery Efficiency and False Negative Rate to Low Concentrations of Two Bacillus anthracis Surrogates on Four Surface Materials

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2014-12-05

    This report describes the experimental design for a laboratory study to quantify the recovery efficiencies and false negative rates of a validated, macrofoam-swab sampling method for low concentrations of Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus (BG) spores on four surface materials (stainless steel, glass, vinyl tile, plastic light cover panel). Two analytical methods (culture and polymerase chain reaction) will be used. Only one previous study has investigated how the false negative rate depends on test factors. The surrogates BAS and BG have not been tested together in the same study previously. Hence, this study will provide for completing gaps in the available information on the performance of macrofoam-swab sampling at low concentrations.

  10. Experimental Design for a Macrofoam Swab Study Relating the Recovery Efficiency and False Negative Rate to Low Concentrations of Two Bacillus anthracis Surrogates on Four Surface Materials

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F.; Hutchison, Janine R.

    2014-04-16

    This report describes the experimental design for a laboratory study to quantify the recovery efficiencies and false negative rates of a validated, macrofoam swab sampling method for low concentrations of Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus (BG) spores on four surface materials (stainless steel, glass, vinyl tile, plastic light cover panel). Two analytical methods (plating/counting and polymerase chain reaction) will be used. Only one previous study has investigated false negative as a function of affecting test factors. The surrogates BAS and BG have not been tested together in the same study previously. Hence, this study will provide for completing gaps in the available information on the performance of macrofoam swab sampling at low concentrations.

  11. Bacillus Coagulans

    Science.gov (United States)

    ... and, as a result, is often misclassified as lactic acid bacteria such as lactobacillus. In fact, some commercial products ... sporogenes or "spore-forming lactic acid bacterium." Unlike lactic acid bacteria such as lactobacillus or bifidobacteria, Bacillus coagulans forms ...

  12. 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.

  13. Base substitution spectra of nalidixylate resistant mutations induced by monochromatic soft X and 60Co γ-rays in bacillus subtilis spores

    International Nuclear Information System (INIS)

    Takahashi, Nobuhiro; Hieda, Kotaro; Morohoshi, Fumiko; Munakata, Nobuo

    1999-01-01

    Bacillus subtilis spores were exposed to three types of photons, monochromatic soft X-rays with the energy corresponding to the absorption peak of phosphorus K-shell electron (2,153 eV) and with the slightly lower energy (2,147 eV), and 60 Co γ-rays. From the irradiated spores, 233 mutants exhibiting nalidixic acid resistance were isolated, and together with 94 spontaneous mutants, the sequence changes in the 5'-terminal region of the gyrA gene coding for DNA gyrase subunit A were determined. Among eighteen alleles of the gyrA mutations, eight were single-base substitutions, nine were tandem double-base substitutions, and one was a double substitution skipping a middle base pair. About 6% of the radiation-induced mutations were tandem double-base substitutions, whereas none was observed among the spontaneous ones. Among spontaneous mutations, A:T and G:C pairs were equally subjected to mutations, whereas the substitutions from G:C pairs and those to A:T pairs predominated among those induced with soft X-rays. The peak-energy X-rays were more effective in killing and causing mutations than the low-energy X-rays, however, there seemed no base-change events uniquely attributable to phosphorus K-shell absorption. (author)

  14. 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)

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

    2012-01-01

    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. PMID:23001669

  15. 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

  16. 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

  17. 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

  18. Decontamination Efficacy of Three Commercial-Off-The-Shelf (COTS) Sporicidal Disinfectants on Medium-Sized Panels Contaminated with Surrogate Spores of Bacillus anthracis

    Science.gov (United States)

    Sabol, Jonathan P.

    2014-01-01

    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. PMID:24940605

  19. 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.

  20. 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.

    2003-01-01

    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°C and 80% relative humidity), the zeolite coating produced approximately 3 log10 inactivation of vegetative cells within a 5- to 24-h period, but viability of spores of the three species was not significantly affected. PMID:12839825

  1. A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Fernando H Ramírez-Guadiana

    2017-09-01

    Full Text Available One of the hallmarks of bacterial endospore formation is the accumulation of high concentrations of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA in the developing spore. This small molecule comprises 5-15% of the dry weight of dormant spores and plays a central role in resistance to both wet heat and desiccation. DPA is synthesized in the mother cell at a late stage in sporulation and must be translocated across two membranes (the inner and outer forespore membranes that separate the mother cell and forespore. The enzymes that synthesize DPA and the proteins required to translocate it across the inner forespore membrane were identified over two decades ago but the factors that transport DPA across the outer forespore membrane have remained mysterious. Here, we report that SpoVV (formerly YlbJ is the missing DPA transporter. SpoVV is produced in the mother cell during the morphological process of engulfment and specifically localizes in the outer forespore membrane. Sporulating cells lacking SpoVV produce spores with low levels of DPA and cells engineered to express SpoVV and the DPA synthase during vegetative growth accumulate high levels of DPA in the culture medium. SpoVV resembles concentrative nucleoside transporters and mutagenesis of residues predicted to form the substrate-binding pocket supports the idea that SpoVV has a similar structure and could therefore function similarly. These findings provide a simple two-step transport mechanism by which the mother cell nurtures the developing spore. DPA produced in the mother cell is first translocated into the intermembrane space by SpoVV and is then imported into the forespore by the SpoVA complex. This pathway is likely to be broadly conserved as DPA synthase, SpoVV, and SpoVA proteins can be found in virtually all endospore forming bacteria.

  2. A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis.

    Science.gov (United States)

    Ramírez-Guadiana, Fernando H; Meeske, Alexander J; Rodrigues, Christopher D A; Barajas-Ornelas, Rocío Del Carmen; Kruse, Andrew C; Rudner, David Z

    2017-09-01

    One of the hallmarks of bacterial endospore formation is the accumulation of high concentrations of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA) in the developing spore. This small molecule comprises 5-15% of the dry weight of dormant spores and plays a central role in resistance to both wet heat and desiccation. DPA is synthesized in the mother cell at a late stage in sporulation and must be translocated across two membranes (the inner and outer forespore membranes) that separate the mother cell and forespore. The enzymes that synthesize DPA and the proteins required to translocate it across the inner forespore membrane were identified over two decades ago but the factors that transport DPA across the outer forespore membrane have remained mysterious. Here, we report that SpoVV (formerly YlbJ) is the missing DPA transporter. SpoVV is produced in the mother cell during the morphological process of engulfment and specifically localizes in the outer forespore membrane. Sporulating cells lacking SpoVV produce spores with low levels of DPA and cells engineered to express SpoVV and the DPA synthase during vegetative growth accumulate high levels of DPA in the culture medium. SpoVV resembles concentrative nucleoside transporters and mutagenesis of residues predicted to form the substrate-binding pocket supports the idea that SpoVV has a similar structure and could therefore function similarly. These findings provide a simple two-step transport mechanism by which the mother cell nurtures the developing spore. DPA produced in the mother cell is first translocated into the intermembrane space by SpoVV and is then imported into the forespore by the SpoVA complex. This pathway is likely to be broadly conserved as DPA synthase, SpoVV, and SpoVA proteins can be found in virtually all endospore forming bacteria.

  3. Influence of glutamate on growth, sporulation, and spore properties of Bacillus cereus ATCC 14579 in defined medium

    NARCIS (Netherlands)

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

    2005-01-01

    A chemically defined medium in combination with an airlift fermentor system was used to study the growth and sporulation of Bacillus cereus ATCC 14579. The medium contained six amino acids and lactate as the main carbon sources. The amino acids were depleted during exponential growth, while lactate

  4. Recovery of Bacillus thuringiensis and related spore-forming bacteria from soil after application for gypsy moth control

    Science.gov (United States)

    Phyllis A.W. Martin; Elizabeth A. Mongeon; Michael B. Blackburn; Dawn E. Gundersen-Rindal

    2011-01-01

    Bacillus thuringiensis Berliner (Bt) has been applied for gypsy moth (Lymantria dispar L.) control in forests in the northeastern U.S. for many years. The subspecies of Bt that is used (urstaki) is not common in U.S. soil. We attempted to recover Bt from...

  5. Survival and Germination of Bacillus cereus Spores without Outgrowth or Enterotoxin Production during In Vitro Simulation of Gastrointestinal Transit

    OpenAIRE

    Ceuppens, Siele; Uyttendaele, Mieke; Drieskens, Katrien; Heyndrickx, Marc; Rajkovic, Andreja; Boon, Nico; Van de Wiele, Tom

    2012-01-01

    To study the gastrointestinal survival and enterotoxin production of the food-borne pathogen Bacillus cereus, an in vitro simulation experiment was developed to mimic gastrointestinal passage in 5 phases: (i) the mouth, (ii) the stomach, with gradual pH decrease and fractional emptying, (iii) the duodenum, with high concentrations of bile and digestive enzymes, (iv) dialysis to ensure bile reabsorption, and (v) the ileum, with competing human intestinal bacteria. Four different B. cereus stra...

  6. Thermal inactivation of Bacillus cereus spores affected by the solutes used to control water activity of the heating medium.

    Science.gov (United States)

    Mazas, M; Martínez, S; López, M; Alvarez, A B; Martin, R

    1999-12-01

    The heat resistance of B. cereus spores (ATCC 7004, 4342 and 9818) over a wide temperature range (92-125 degrees C) in aqueous solutions of NaCl, LiCl, sucrose and glycerol at different water activities (1.00-0.71) was investigated. Sodium chloride in the heating medium tended to protect the spores of B. cereus against heat. The z-values increased significantly (P 0.87 M), the D-values showed an increase, although only those obtained for strain 4342 in sucrose solutions 2.22 M were higher than those found in pure water. The z-values were significantly higher (P < 0.05) when sucrose was added at concentrations above 1.42 M, except for strain 4342. When a(w) was lowered from 0.96 to 0.71 with glycerol, D-values obtained gradually increased, about 30, 50 and 60 fold for 4342, 7004 and 9818 strains, respectively. No significant effect on z-values were detected.

  7. [Development and comparative evaluation of up-converting phosphor technology based lateral flow assay for rapid detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp].

    Science.gov (United States)

    Li, Chunfeng; Zhang, Pingping; Wang, Xiaoying; Liu, Xiao; Zhao, Yong; Sun, Chongyun; Wang, Chengbin; Yang, Ruifu; Zhou, Lei

    2015-01-01

    To develop an up-converting phosphor technology based lateral flow (UPT-LF) assay for rapid and quantitative detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp.and make the comparison with BioThreat Alert (BTA) test strips (Tetracore Inc., USA). Using up-converting phosphor nano-particles (UCP-NPs) as the bio-marker, three double-antibody-sandwich model based UPT-LF strips including Plague-UPT-LF, Anthrax-UPT-LF, Brucella-UPT-LF were prepared and its sensitivity, accuracy, linearity and specificity were determined by detecting 10(10), 10(9), 10(8), 10(7), 10(6), 10(5) and 0 CFU/ml series of concentrations of Y.pestis, B.anthracis, Brucella standards and other 27 kinds of 10(9) CFU/ml series of contrations of bacteria strains.Furthermore, the speed, sensitivity and accuracy of bacteria standards and simulated sample detection were compared between UPT-LF and BTA system. The detection limit of Plague-UPT-LF, Anthrax-UPT-LF and Brucella-LF was 10(5) CFU/ml. The CV of series of bacteria concentrations was ≤ 15%, and the r between lg (T/C-cut-off) and lg (concentration) was 0.996,0.998 and 0.999 (F values were 1 647.57, 743.51 and 1 822.17. All the P values were Brucella-LF were excellent, while that of Anthrax-UPT-LF was a little bit regretful because of non-specific reaction with two isolates of B. subtilis and one B.cereus. On-site evaluation showed the detection time of UPT-LF for all Y.pestis, B.anthracis spore and Brucella spp.was 33, 36 and 37 min, while BTA was 115, 115 and 111 min, which revealed the higher detection speed and sensitivity of UPT-LF comparing with BTA. The negative rate of two methods for blank standard was both 5/5, the sensitivity of UPT-LF for Y.pestis,B.anthracis spore and Brucella spp. was all 10(5) CFU/ml, then BTA was 10(6), 10(6) and 10(5) CFU/ml, respectively. The detection rate of UPT-LF for all three bacteria analog positive samples was 16/16, while BTA for B.anthracis was 7/16 only. The good performance

  8. YtkD and MutT Protect Vegetative Cells but Not Spores of Bacillus subtilis from Oxidative Stress

    OpenAIRE

    Castellanos-Juárez, Francisco X.; Álvarez-Álvarez, Carlos; Yasbin, Ronald E.; Setlow, Barbara; Setlow, Peter; Pedraza-Reyes, Mario

    2006-01-01

    ytkD and mutT of Bacillus subtilis encode potential 8-oxo-dGTPases that can prevent the mutagenic effects of 8-oxo-dGTP. Loss of YtkD but not of MutT increased the spontaneous mutation frequency of growing cells. However, cells lacking both YtkD and MutT had a higher spontaneous mutation frequency than cells lacking YtkD. Loss of either YtkD or MutT sensitized growing cells to hydrogen peroxide (H2O2) and t-butylhydroperoxide (t-BHP), and the lack of both proteins sensitized growing cells to ...

  9. 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.

  10. False Negative Rates of a Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates via Real-Time PCR

    Energy Technology Data Exchange (ETDEWEB)

    Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Amidan, Brett G. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sydor, Michael A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Kaiser, Brooke L.D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-06-01

    Surface sampling for Bacillus anthracis spores has traditionally relied on detection via bacterial cultivation methods. Although effective, this approach does not provide the level of organism specificity that can be gained through molecular techniques. False negative rates (FNR) and limits of detection (LOD) were determined for two B. anthracis surrogates with modified rapid viability-polymerase chain reaction (mRV-PCR) following macrofoam-swab sampling. This study was conducted in parallel with a previously reported study that analyzed spores using a plate-culture method. B. anthracis Sterne (BAS) or B. atrophaeus Nakamura (BG) spores were deposited onto four surface materials (glass, stainless steel, vinyl tile, and plastic) at nine target concentrations (2 to 500 spores/coupon; 0.078 to 19.375 colony-forming units [CFU] per cm2). Mean FNR values for mRV-PCR analysis ranged from 0 to 0.917 for BAS and 0 to 0.875 for BG and increased as spore concentration decreased (over the concentrations investigated) for each surface material. FNRs based on mRV-PCR data were not statistically different for BAS and BG, but were significantly lower for glass than for vinyl tile. FNRs also tended to be lower for the mRV-PCR method compared to the culture method. The mRV-PCR LOD95 was lowest for glass (0.429 CFU/cm2 with BAS and 0.341 CFU/cm2 with BG) and highest for vinyl tile (0.919 CFU/cm2 with BAS and 0.917 CFU/cm2 with BG). These mRV-PCR LOD95 values were lower than the culture values (BAS: 0.678 to 1.023 CFU/cm2 and BG: 0.820 to 1.489 CFU/cm2). The FNR and LOD95 values reported in this work provide guidance for environmental sampling of Bacillus spores at low concentrations.

  11. 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

  12. The effect of Ca2+ ions and ionic strength on Mn(II) oxidation by spores of the marine Bacillus sp. SG-1

    Science.gov (United States)

    Toyoda, Kazuhiro; Tebo, Bradley M.

    2013-01-01

    Manganese(IV) oxides, believed to form primarily through microbial activities, are extremely important mineral phases in marine environments where they scavenge a variety of trace elements and thereby control their distributions. The presence of various ions common in seawater are known to influence Mn oxide mineralogy yet little is known about the effect of these ions on the kinetics of bacterial Mn(II) oxidation and Mn oxide formation. We examined factors affecting bacterial Mn(II) oxidation by spores of the marine Bacillus sp. strain SG-1 in natural and artificial seawater of varying ionic conditions. Ca2+ concentration dramatically affected Mn(II) oxidation, while Mg2+, Sr2+, K+, Na+ and NO3- ions had no effect. The rate of Mn(II) oxidation at 10 mM Ca2+ (seawater composition) was four or five times that without Ca2+. The relationship between Ca2+ content and oxidation rate demonstrates that the equilibrium constant is small (on the order of 0.1) and the binding coefficient is 0.5. The pH optimum for Mn(II) oxidation changed depending on the amount of Ca2+ present, suggesting that Ca2+ exerts a direct effect on the enzyme perhaps as a stabilizing bridge between polypeptide components. We also examined the effect of varying concentrations of NaCl or KNO3 (0-2000 mM) on the kinetics of Mn(II) oxidation in solutions containing 10 mM Ca2+. Mn(II) oxidation was unaffected by changes in ionic strength (I) below 0.2, but it was inhibited by increasing salt concentrations above this value. Our results suggest that the critical coagulation concentration is around 200 mM of salt (I = ca. 0.2), and that the ionic strength of seawater (I > 0.2) accelerates the precipitation of Mn oxides around the spores. Under these conditions, the aggregation of Mn oxides reduces the supply of dissolved O2 and/or Mn2+ and inhibits the Mn(II) → Mn(III) step controlling the enzymatic oxidation of Mn(II). Our results suggest that the hardness and ionic strength of the aquatic environment

  13. Three non-aspartate amino acid mutations in the ComA Response regulator receiver motif severely decrease surfactin production, competence development and spore formation in Bacillus subtilis.

    Science.gov (United States)

    Wang, Xiaoyu; Luo, Chuping; Liu, Youzhou; Nie, Yafeng; Liu, Yongfeng; Zhang, Rongsheng; Chen, Zhiyi

    2010-02-01

    Bacillus subtilis strains produce a broad spectrum of bioactive peptides. The lipopeptide surfactin belongs to one wellknown class, which includes amphiphilic membrane-active biosurfactants and peptide antibiotics. Both the srfA promoter and the ComP-ComA signal transduction system are an important part of the factor that results in the production of surfactin. Bs-M49, obtained by means of low-energy ion implantation in wild-type Bs-916, produced significantly lower levels of surfactin, and had no obvious effects against R. solani. Occasionally, we found strain Bs- M49 decreased spore formation and the development of competence. Blast comparison of the sequences from Bs- 916 and M49 indicate that there is no difference in the srfA operon promoter PsrfA, but there are differences in the coding sequence of the comA gene. These differences result in three missense mutations within the M49 ComA protein. RT-PCR analyses results showed that the expression levels of selected genes involved in competence and sporulation in both the wild-type Bs-916 and mutant M49 strains were significantly different. When we integrated the comA ORF into the chromosome of M49 at the amyE locus, M49 restored hemolytic activity and antifungal activity. Then, HPLC analyses results also showed the comA-complemented strain had a similar ability to produce surfactin with wild-type strain Bs-916. These data suggested that the mutation of three key amino acids in ComA greatly affected the biological activity of Bacillus subtilis. ComA protein 3D structure prediction and motif search prediction indicated that ComA has two obvious motifs common to response regulator proteins, which are the Nterminal response regulator receiver motif and the Cterminal helix-turn-helix motif. The three residues in the ComA N-terminal portion may be involved in phosphorylation activation mechanism. These structural prediction results implicate that three mutated residues in the ComA protein may play an important role in

  14. A comparative study of the disinfection efficacy of H2O2/ferrate and UV/H2O2/ferrate processes on inactivation of Bacillus subtilis spores by response surface methodology for modeling and optimization.

    Science.gov (United States)

    Matin, Atiyeh Rajabi; Yousefzadeh, Samira; Ahmadi, Ehsan; Mahvi, Amirhossein; Alimohammadi, Mahmood; Aslani, Hassan; Nabizadeh, Ramin

    2018-04-03

    Although chlorination can inactivate most of the microorganisms in water but protozoan parasites like C. parvum oocysts and Giardia cysts can resist against it. Therefore, many researches have been conducted to find a novel method for water disinfection. Present study evaluated the synergistic effect of H2O2 and ferrate followed by UV radiation to inactivate Bacillus subtilis spores as surrogate microorganisms. Response surface methodology(RSM) was employed for the optimization for UV/H2O2/ferrate and H2O2/ferrate processes. By using central composite design(CCD), the effect of three main parameters including time, hydrogen peroxide, and ferrate concentrations was examined on process performance. The results showed that the combination of UV, H2O2 and ferrate was the most effective disinfection process in compare with when H2O2 and ferrate were used. This study indicated that by UV/H2O2/ferrate, about 5.2 log reductions of B. subtilis spores was inactivated at 9299 mg/l of H2O2 and 0.4 mg/l of ferrate concentrations after 57 min of contact time which was the optimum condition, but H2O2/ferrate can inactivate B. subtilis spores about 4.7 logs compare to the other process. Therefore, the results of this research demonstrated that UV/H2O2 /ferrate process is a promising process for spore inactivation and water disinfection. Copyright © 2018 Elsevier Ltd. All rights reserved.

  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.

    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.

  16. Esterilização por óxido de etileno: I. Influência do meio de esporulação na resistência dos esporos de Bacillus subtilis var. niger Ethylene oxide sterilization: I. The influence of sporulation medium in the resistance of the spores of Bacillus subtilis var. niger

    Directory of Open Access Journals (Sweden)

    Terezinha de Jesus A. Pinto

    1992-12-01

    Full Text Available Tendo por meta a padronização das variáveis influenciando a resistência de esporos empregados no controle do processo esterilizante por óxido de etileno, foram obtidos esporos de Bacillus subtilis var. niger, em meio sólido e líquido sintético de esporulação. Tais esporos, após padronização quantitativa dos 12 lotes obtidos, foram submetidos a exposições subletais como bioindicadores, tendo o papel como suporte. Construiu-se, então, a curva de letalidade característica de cada lote. A análise estatística empregada não evidenciou diferenças entre resistência dos 10 lotes obtidos em meio sólido e os 2 em meio líquido sintético, ressaltando-se a vantagem quanto ao rendimento que caracterizou a primeira metodologia.Some elements influencing the resistance of spores used in ethylene oxide sterilization process control are standardized. Spores of Bacillus subtilis var. niger were produced in chemically defined liquid and solid sporulation media to a total of 12 lots; after standardization of the number of spores, they were challenged by sub-lethal cycles, followed by a lethality study. According to the statistical model applied, there were no differences between the resistance of spores produced in chemically defined liquid and those produced in solid sporulation media. The advantage of the solid sporulation media consists in the larger production of spores.

  17. Effect of individual or combined treatment by γ-irradiation or temperature (high or low) on bacillus subtilis spores and its application for sterilization of ground beef

    International Nuclear Information System (INIS)

    El-Zawahry, Y.A.; Mostafa, S.A.; Awny, N.M.

    1986-01-01

    The combination of two lethal agents such as irradiation and temperature (high or sub zero) resulted in synergistic death or B. subtilis spores (as indicated by decrease in the thermal D-value). The extent of this synergism in killing a spore population depended mainly on the sequence on application of the two physical agents. Irradiation-temperature (high or sub zero) sequence killed more but injured less B. subtilis spores than temperature irradiation sequence or irradiation and temperature applied separately. Storage at -20 0 C killed more spores than storage at -2 0 C if carried after irradiation, while the reverse was true of storage was prior irradiation. An irradiation dose of 8 KGY followed by thermal exposure to 70 0 C for 1 hr is suggested for the sterilization of ground beef. Irradiation induced certain quantitative changes on the amino-N, protein-N, RNA and DNA of the first subcultures of irradiated spores with stimulatory effect at low irradiation doses and inhibitory effect at the high irradiation doses. This might explain the increased sensitivity of irradiated spores to subsequent exposure to unfavourable temperature (high or sub zero). Exposure of B. subtilis spore to 70 0 C induced a stimulation in the amino- and protein-N of the resulting cells while exposure to 80 0 C resulted in a significant decrease in the amino-N. The protein-N remained more or less the same

  18. Impact of temperature, nutrients, pH and cold storage on the germination, growth and resistance of Bacillus cereus spores in egg white.

    Science.gov (United States)

    Soni, Aswathi; Oey, Indrawati; Silcock, Patrick; Bremer, Phil J

    2018-04-01

    B. cereus spores are a concern to the food industry, especially to the producers of heat sensitive food products like egg white or precooked and stored food such as fried rice. This study investigated the impact of nutrients, temperature (4, 8, 15 and 25 °C), pH (4, 5, 7 and 9), and cold storage on the germination, growth and resistance of B. cereus spores. In egg white held at 4 °C for 12 days spore germination was not apparent, however the addition of egg yolk (5%) resulted in a 2 Log colony forming units (CFU)/mL increase in vegetative cells (p egg white did not induce germination unless the spores were simultaneously heat activated at 70 °C for 30 min. On incubation at 15 or 25 °C in egg white, spore germination increased by 3.0 Log and 3.7 Log CFU/mL on day 12. The presence of 5% yolk further enhanced germination and subsequent sporulation during storage at 15 and 25 °C. Acidification (pH 4) of 10% egg white solution prevented germination at 4, 8, 15 and 25 °C. Spores held at 4 °C for 6 days in phosphate buffer (50 mM, pH 4) had visible deformations on their surface (scanning electron microscopy) and a significant reduction in D 88, and D 92 values of 13.9 and 8.2 min respectively. A better understanding of how spores sense and respond to changing environmental conditions will help in the development of processing strategies, involving multiple hurdles to ensure the prevention of germination and subsequent toxin production. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. 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

  20. 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.

    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

  1. Photometric immersion refractometry of bacterial spores.

    Science.gov (United States)

    Gerhardt, P; Beaman, T C; Corner, T R; Greenamyre, J T; Tisa, L S

    1982-01-01

    Photometric immersion refractometry was used to determine the average apparent refractive index (n) of five types of dormant Bacillus spores representing a 600-fold range in moist-heat resistance determined as a D100 value. The n of a spore type increased as the molecular size of various immersion solutes decreased. For comparison of the spore types, the n of the entire spore and of the isolated integument was determined by use of bovine serum albumin, which is excluded from permeating into them. The n of the sporoplast (the structures bounded by the outer pericortex membrane) was determined by use of glucose, which was shown to permeate into the spore only as deeply as the pericortex membrane. Among the various spore types, an exponential increase in the heat resistance correlated with the n of the entire spore and of the sporoplast, but not of the isolated perisporoplast integument. Correlation of the n with the solids content of the entire spore provided a method of experimentally obtaining the refractive index increment (dn/dc), which was constant for the various spore types and enables the calculation of solids and water content from an n. Altogether, the results showed that the total water content is distributed unequally within the dormant spore, with less water in the sporoplast than in the perisporoplast integument, and that the sporoplast becomes more refractile and therefore more dehydrated as the heat resistance becomes greater among the various spore types. PMID:6802796

  2. 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.

  3. Oral delivery of Bacillus subtilis spores expressing cysteine protease of Clonorchis sinensis to grass carp (Ctenopharyngodon idellus): Induces immune responses and has no damage on liver and intestine function.

    Science.gov (United States)

    Tang, Zeli; Sun, Hengchang; Chen, TingJin; Lin, Zhipeng; Jiang, Hongye; Zhou, Xinyi; Shi, Cunbin; Pan, Houjun; Chang, Ouqin; Ren, Pengli; Yu, Jinyun; Li, Xuerong; Xu, Jin; Huang, Yan; Yu, Xinbing

    2017-05-01

    Clonorchis sinensis (C. sinensis) is a fish-borne trematode. Human can be infected by ingestion of C. sinensis metacercariae parasitized in grass carp (Ctenopharyngodon idella). For induction of effective oral immune responses, spores of Bacillus subtilis (B. subtilis) WB600 were utilized as vehicle to delivery CsCP (cysteine protease of C. sinensis) cooperated with CotC (B.s-CotC-CP), one of coat proteins, to the gastrointestinal tract. After routine culture of 8-12 h in LB medium, B. subtilis containing CotC-CsCP was transferred into the sporulation culture medium. SDS-PAGE, western blotting and the growth curve indicated that the best sporulation time of recombinant WB600 was 24-30 h at 37 °C with continuous shaking (250 rpm). Grass carp were fed with three levels of B.s-CotC-CP (1 × 10 6 , 1 × 10 7 , and 1 × 10 8  CFU g -1 ) incorporated in the basal pellets diet. The commercial pellets or supplemented with spores just expressing CotC (1 × 10 7  CFU g -1 ) were served as control diet. Our results showed that grass carp orally immunized with the feed-based B.s-CotC-CP developed a strong specific immune response with significantly (P sinensis in fish body. Therefore, this study demonstrated that the feed-based recombinant spores could trigger high levels of mucosal and humoral immunity, and would be a promising candidate vaccine against C. sinensis metacercariae formation in freshwater fish. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Comparative analysis of Bacillus subtilis spores and monophosphoryl lipid A as adjuvants of protein-based mycobacterium tuberculosis-based vaccines: partial requirement for interleukin-17a for induction of protective immunity.

    Science.gov (United States)

    Esparza-Gonzalez, Sandra C; Troy, Amber R; Izzo, Angelo A

    2014-04-01

    The development of adjuvants for vaccines has become an important area of research as the number of protein-based vaccines against infectious pathogens increases. Currently, there are a number of adjuvant-based Mycobacterium tuberculosis vaccines in clinical trials that have shown efficacy in animal models. Despite these novel adjuvants, there is still a need to design new and more versatile adjuvants that have minimal adverse side effects but produce robust long-lasting adaptive immune responses. To this end, we hypothesized that Bacillus subtilis spores may provide the appropriate innate signals that are required to generate such vaccine-mediated responses, which would be sufficient to reduce the mycobacterial burden after infection with M. tuberculosis. In addition, we compared the response generated by B. subtilis spores to that generated by monophosphoryl lipid A (MPL), which has been used extensively to test tuberculosis vaccines. The well-characterized, 6-kDa early secretory antigenic target of M. tuberculosis (ESAT-6; Rv3875) was used as a test antigen to determine the T cell activation potential of each adjuvant. Inoculated into mice, B. subtilis spores induced a strong proinflammatory response and Th1 immunity, similar to MPL; however, unlike MPL formulated with dimethyldioctadecylammonium (DDA) bromide, it failed to induce significant levels of interleukin-17A (IL-17A) and was unable to significantly reduce the mycobacterial burden after pulmonary infection with M. tuberculosis. Further analysis of the activity of MPL-DDA suggested that IL-17A was required for protective immunity. Taken together, the data emphasize the requirement for a network of cytokines that are essential for protective immunity.

  5. Decontamination of B. globigii spores from drinking water infrastructure using disinfectants

    Data.gov (United States)

    U.S. Environmental Protection Agency — Decontamination of Bacillus spores adhered to common drinking water infrastructure surfaces was evaluated using a variety of disinfectants. Corroded iron and...

  6. 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

    The effect of modified atmosphere packaging (MAP) on the germination and growth of toxin producingpsychrotolerant Bacillus spp is not well described. A model agar system mimicking a cooked meat product wasused in initial experiments. Incubation at refrigeration temperature of 8 °C for 5 weeks of 26...... 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...

  7. Ultraviolet-Resistant Bacterial Spores

    Science.gov (United States)

    Venkateswaran, Kasthuri; Newcombe, David; LaDuc, Myron T.; Osman, Shariff R.

    2007-01-01

    A document summarizes a study in which it was found that spores of the SAFR-032 strain of Bacillus pumilus can survive doses of ultraviolet (UV) radiation, radiation, and hydrogen peroxide in proportions much greater than those of other bacteria. The study was part of a continuing effort to understand the survivability of bacteria under harsh conditions and develop means of sterilizing spacecraft to prevent biocontamination of Mars that could interfere with the search for life there.

  8. False Negative Rates of a Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates via Real-Time PCR

    Energy Technology Data Exchange (ETDEWEB)

    Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Amidan, Brett G. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sydor, Michael A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Deatherage Kaiser, Brooke L [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2015-05-01

    Surface sampling for Bacillus anthracis spores has traditionally relied on detection via bacterial cultivation methods. Although effective, this approach does not provide the level of organism specificity that can be gained through molecular techniques. False negative rates (FNR) and limits of detection (LOD) were determined for two B. anthracis surrogates with modified rapid viability-polymerase chain reaction (mRV-PCR) following macrofoam-swab sampling. This study was conducted in parallel with a previously reported study that analyzed spores using a plate-culture method. B. anthracis Sterne (BAS) or B. atrophaeus Nakamura (BG) spores were deposited onto four surface materials (glass, stainless steel, vinyl tile, and plastic) at nine target concentrations (2 to 500 spores/coupon; 0.078 to 19.375 colony-forming units [CFU] per cm²). Mean FNR values for mRV-PCR analysis ranged from 0 to 0.917 for BAS and 0 to 0.875 for BG and increased as spore concentration decreased (over the concentrations investigated) for each surface material. FNRs based on mRV-PCR data were not statistically different for BAS and BG, but were significantly lower for glass than for vinyl tile. FNRs also tended to be lower for the mRV-PCR method compared to the culture method. The mRV-PCR LOD₉₅ was lowest for glass (0.429 CFU/cm² with BAS and 0.341 CFU/cm² with BG) and highest for vinyl tile (0.919 CFU/cm² with BAS and 0.917 CFU/cm² with BG). These mRV-PCR LOD₉₅ values were lower than the culture values (BAS: 0.678 to 1.023 CFU/cm² and BG: 0.820 to 1.489 CFU/cm²). The FNR and LOD₉₅ values reported in this work provide guidance for environmental sampling of Bacillus spores at low concentrations.

  9. Detecting bacterial spores in soup manufacturing

    NARCIS (Netherlands)

    van Zuijlen, A.C.M.; Oomes, S.J.C.M.; Vos, P.; Brul, S.

    2009-01-01

    Spores from mesophilic aerobic sporeforming bacteria (Bacillus) are sometimes able to survive the thermal process of commercial sterile products and sporadically cause spoilage or food poisoning. Because of an increasing demand for more fresh products, ideally the processing temperatures should be

  10. Modeling to control spores in raw milk

    NARCIS (Netherlands)

    Vissers, M.

    2007-01-01

    A modeling approach was used to identify measures at the farm that reduce transmission of microorganisms to raw milk. Butyric acid bacteria (BAB) and Bacillus cereus were used as case-studies. Minimizing the concentration of BAB spores in raw milk is important to prevent late-blowing of Gouda-type

  11. Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74∆sp chitinase inclusions, Cry crystals and spores for applied use

    Science.gov (United States)

    2014-01-01

    Background The endochitinase ChiA74 is a soluble secreted enzyme produced by Bacillus thuringiensis that synergizes the entomotoxigenecity of Cry proteins that accumulate as intracellular crystalline inclusion during sporulation. The purpose of this study was to produce alkaline-soluble ChiA74∆sp inclusions in B. thuringiensis, and to determine its effect on Cry crystal production, sporulation and toxicity to an important agronomical insect, Manduca sexta. To this end we deleted the secretion signal peptide-coding sequence of chiA74 (i.e. chiA74∆sp) and expressed it under its native promoter (pEHchiA74∆sp) or strong chimeric sporulation-dependent cytA-p/STAB-SD promoter (pEBchiA74∆sp) in Escherichia coli, acrystalliferous B. thuringiensis (4Q7) and B. thuringiensis HD1. Results Based on mRNA analyses, up to ~9-fold increase in expression of chiA74∆sp was observed using the cytA-p/STAB-SD promoter. ChiA74∆sp (~70 kDa) formed intracellular inclusions that frequently accumulated at the poles of cells. ChiA74∆sp inclusions were dissolved in alkali and reducing conditions, similar to Cry crystals, and retained its activity in a wide range of pH (5 to 9), but showed a drastic reduction (~70%) at pH 10. Chitinase activity of E. coli-pEHchiA74∆sp was ~150 mU/mL, and in E. coli-pEBchiA74∆sp, 250 mU/mL. 4Q7-pEBchiA74∆sp and 4Q7-pEHchiA74∆sp had activities of ~127 mU/mL and ~41 mU/mL, respectively. The endochitinase activity in HD1-pEBchiA74∆sp increased 42x when compared to parental HD1 strain. HD1-pEBchiA74∆sp and HD1 harbored typical bipyramidal Cry inclusions, but crystals in the recombinant were ~30% smaller. Additionally, a 3x increase in the number of viable spores was observed in cultures of the recombinant strain when compared to HD1. Bioassays against first instar larvae of M. sexta with spore-crystals of HD1 or spore-crystal-ChiA74∆sp inclusions of HD1-pEBchiA74∆sp showed LC50s of 67.30 ng/cm2 and 41.45 ng/cm2, respectively

  12. [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.

  13. Lyophilized spore dispenser

    Science.gov (United States)

    Jessup, A. D. (Inventor)

    1973-01-01

    A lyophilized spore dispenser is provided which produces a finely divided, monoparticulate cloud of bacterial spores. The spores are contained within a tightly sealed chamber, and a turbulator orifice connected to an air supply source provides a jet of air which stirs up the spores and causes the spores to be suspended in eddy currents within the chamber. This air jet also produces a positive pressure within the chamber which forces the spores out of an injection orifice.

  14. Imaging bacterial spores by soft-x-ray microscopy

    International Nuclear Information System (INIS)

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

    1997-01-01

    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

  15. 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.

  16. Recovery Efficiency, False Negative Rate, and Limit of Detection Performance of a Validated Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Kaiser, Brooke L. D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Amidan, Brett G. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sydor, Michael A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Barrett, Christopher A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-06-16

    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, 40.2% with BG) and the highest for glass (92.8% with BAS, 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG, with values increasing as concentration decreased in the range tested (0.078 to 19.375 CFU/cm2, where CFU denotes ‘colony forming units’). 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 are discussed in a separate report.

  17. Recovery Efficiency, False Negative Rate, and Limit of Detection Performance of a Validated Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Deatherage Kaiser, Brooke L [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Amidan, Brett G. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sydor, Michael A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Barrett, Christopher A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2015-03-31

    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, 40.2% with BG) and the highest for glass (92.8% with BAS, 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG, with values increasing as concentration decreased in the range tested (0.078 to 19.375 CFU/cm2, where CFU denotes ‘colony forming units’). 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 report.

  18. 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.

  19. 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 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).

  20. Effect of Feeding Bacillus subtilis Spores to Broilers Challenged with Salmonella enterica serovar Heidelberg Brazilian Strain UFPR1 on Performance, Immune Response, and Gut Health

    Directory of Open Access Journals (Sweden)

    Ricardo Mitsuo Hayashi

    2018-02-01

    Full Text Available Salmonellosis is a poultry industry and public health concern worldwide. Recently, Salmonella enterica serovar Heidelberg (SH has been reported in broilers in Brazil. The effect of feeding a blend of three strains of Bacillus subtilis (PRO was studied in broilers orally challenged (107 CFU/chick or not with a SH isolated in south of Brazil (UFPR1 strain. Twelve male Cobb 500 broilers per pen were randomly assigned to six treatments in a 3 × 2 factorial experiment where PRO was added at 0, 250, or 500 g/ton of broiler feed and fed to either SH-challenged (SH Control, SH + PRO 250, and SH + PRO 500 or non-challenged birds (Control, PRO 250, and PRO 500. Broiler performance, histologic alterations in intestinal morphology, Salmonella quantification and immune cells counts in liver (macrophages, T CD4+ and T CD8+ were analyzed. Changes in the intestinal microbiota of broilers were also studied by metagenomics for Control, SH Control, SH + PRO 250, and SH + PRO 500 only. Feeding PRO at 250 or 500 g/ton reduced SH counts and incidence in liver and cecum at 21 days of age. It was observed that PRO groups increased the macrophage mobilization to the liver in SH-challenged birds (P < 0.05 but reduced these cells in the liver of non-challenged birds, showing an interesting immune cell dynamics effect. PRO at 250 g/ton did not affect gut histology, but improved animal performance (P < 0.05 while PRO at 500/ton did not affect animal performance but increased histologic alteration related to activation of the defense response in the ileum in SH challenged birds compared to control birds (P < 0.05. SH + PRO 500 group presented a more diverse cecal microbiota (Shannon–Wiener index; P < 0.05 compared to Control and SH Control groups; while SH + PRO 250 had greater ileal richness (JackkNife index compared to Control (P < 0.05. PRO was effective in reducing Salmonella colonization in liver and cecum when

  1. Resistance of Bacillus Endospores to Extreme Terrestrial and Extraterrestrial Environments

    Science.gov (United States)

    Nicholson, Wayne L.; Munakata, Nobuo; Horneck, Gerda; Melosh, Henry J.; Setlow, Peter

    2000-01-01

    Endospores of Bacillus spp., especially Bacillus subtilis, have served as experimental models for exploring the molecular mechanisms underlying the incredible longevity of spores and their resistance to environmental insults. In this review we summarize the molecular laboratory model of spore resistance mechanisms and attempt to use the model as a basis for exploration of the resistance of spores to environmental extremes both on Earth and during postulated interplanetary transfer through space as a result of natural impact processes. PMID:10974126

  2. Mucosal delivery of antigens using adsorption to bacterial spores.

    Science.gov (United States)

    Huang, Jen-Min; Hong, Huynh A; Van Tong, Hoang; Hoang, Tran H; Brisson, Alain; Cutting, Simon M

    2010-01-22

    The development of new-generation vaccines has followed a number of strategic avenues including the use of live recombinant bacteria. Of these, the use of genetically engineered bacterial spores has been shown to offer promise as both a mucosal as well as a heat-stable vaccine delivery system. Spores of the genus Bacillus are currently in widespread use as probiotics enabling a case to be made for their safety. In this work we have discovered that the negatively charged and hydrophobic surface layer of spores provides a suitable platform for adsorption of protein antigens. Binding can be promoted under conditions of low pH and requires a potent combination of electrostatic and hydrophobic interactions between spore and immunogen. Using appropriately adsorbed spores we have shown that mice immunised mucosally can be protected against challenge with tetanus toxin, Clostridium perfringens alpha toxin and could survive challenge with anthrax toxin. In some cases protection is actually greater than using a recombinant vaccine. Remarkably, killed or inactivated spores appear equally effective as live spores. The spore appears to present a bound antigen in its native conformation promoting a cellular (T(h)1-biased) response coupled with a strong antibody response. Spores then, should be considered as mucosal adjuvants, most similar to particulate adjuvants, by enhancing responses against soluble antigens. The broad spectrum of immune responses elicited coupled with the attendant benefits of safety suggest that spore adsorption could be appropriate for improving the immunogenicity of some vaccines as well as the delivery of biotherapeutic molecules.

  3. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. 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.

  5. Germination, outgrowth and vegetative growth kinetics of dry heat-treated individual spores ofBacillusspecies.

    Science.gov (United States)

    He, Lin; Chen, Zhan; Wang, Shiwei; Wu, Muying; Setlow, Peter; Li, Yong-Qing

    2018-01-12

    DNA damage kills dry-heated spores of Bacillus subtilis , but dry heat-treatment effects on spore germination and outgrowth have not been studied. This is important, since if dry heat-killed spores germinate and undergo outgrowth, toxic proteins could be synthesized. Here, Raman spectroscopy and differential interference contrast microscopy were used to study germination and outgrowth of individual dry heat-treated B. subtilis and Bacillus megaterium spores. Major findings in this work were as follows. 1) Spores dry heat-treated at 140°C for 20 min nearly all lost viability but retained their Ca 2+ -dipicolinic acid (CaDPA) depot. 2) In most cases, dry heat treatment increased the average times of and variability in all major events in B. subtilis spore germination with nutrient germinants or CaDPA, and one nutrient germination event with B. megaterium spores. 3) B. subtilis spore germination with dodecylamine, which activates spores' CaDPA release channel, was unaffected by dry heat treatment. 4) These results indicate that dry heat treatment likely damages spore proteins important in nutrient germinant recognition and cortex peptidoglycan hydrolysis, but not CaDPA release itself. 5) Analysis of single spores incubated on nutrient-rich agar showed that while dry heat-treated spores that are dead can complete germination, they cannot proceed into outgrowth thus not to vegetative growth. The results of this study provide new information on effects of dry heat on bacterial spores, and indicate that dry heat sterilization regimens should give spores that cannot outgrow and thus cannot synthesize potentially dangerous proteins. IMPORTANCE Much research has shown that high temperature dry heat is a promising means for the inactivation of spores on medical devices and spacecraft decontamination. Dry heat is known to kill Bacillus subtilis spores by DNA damage. However, knowledge about effects of dry heat treatment on spore germination and outgrowth is limited

  6. Evaluation of the Performance of Iodine-Treated Biocide Filters Challenged with Bacterial Spores and Viruses

    National Research Council Canada - National Science Library

    Lee, Jin-Hwa; Wu, Chang-Yu

    2006-01-01

    Filter media coated with a cationic resin in triiodide form were challenged by Bacillus subtilis spores and MS2 bacteriophage aerosols delivered from a Collison nebulizer through air at 35% RH and 23 C...

  7. 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.

  8. 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...

  9. A simple identification method for spore-forming bacteria showing high resistance against γ-rays

    International Nuclear Information System (INIS)

    Koshikawa, Tomihiko; Sone, Koji; Kobayashi, Toshikazu

    1993-01-01

    A simple identification method was developed for spore-forming bacteria which are highly resistant against γ-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 γ-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 γ-rays. The method is specific for B. megaterium, B. thuringiensis and B. pumilus, and highly selective for B. aneurinolyticus and B. cereus. (author)

  10. 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.

  11. Reclassification of Bacillus axarquiensis Ruiz-Garcia et al. 2005 and Bacillus malacitensis Ruiz-Garcia et al. 2005 as later heterotypic synonyms of Bacillus mojavensis Roberts et al. 1994.

    Science.gov (United States)

    Wang, Li-Ting; Lee, Fwu-Ling; Tai, Chun-Ju; Yokota, Akira; Kuo, Hsiao-Ping

    2007-07-01

    The Bacillus subtilis group encompasses the taxa Bacillus subtilis subsp. subtilis, B. licheniformis, B. amyloliquefaciens, B. atrophaeus, B. mojavensis, B. vallismortis, B. subtilis subsp. spizizenii, B. sonorensis, B. velezensis, B. axarquiensis and B. malacitensis. In this study, the taxonomic relatedness between the species B. axarquiensis, B. malacitensis and B. mojavensis was investigated. Sequence analysis of the 16S rRNA gene and the gene for DNA gyrase subunit B (gyrB) confirmed the very high similarities between these three type strains and a reference strain of B. mojavensis (>99 and >97 %, respectively). DNA-DNA hybridization experiments revealed high relatedness values between the type strains of B. axarquiensis, B. malacitensis and B. mojavensis and between these strains and a reference strain of B. mojavensis (83-98 %). Based on these molecular taxonomic data and the lack of phenotypic distinctive characteristics, Bacillus axarquiensis and Bacillus malacitensis should be reclassified as later heterotypic synonyms of Bacillus mojavensis.

  12. The proteome of spore surface layers in food spoiling bacteria

    NARCIS (Netherlands)

    Abhyankar, W.R.

    2014-01-01

    Endospores are dormant, multilayered, highly resistant cellular structures formed in response to stress by certain bacteria belonging to the genera Bacillus, Clostridium and other related organisms. In presence of nutrients and favorable conditions spores germinate and grow out as normal vegetative

  13. Spore Proteomics: The Past, Present and the Future

    NARCIS (Netherlands)

    Abhyankar, W.; de Koning, L.J.; Brul, S.; de Koster, C.G.

    2014-01-01

    Endospores are metabolically dormant, multi-layered cellular structures formed by Gram positive bacteria belonging to the genera Bacillus, Clostridium and related organisms. Their external layers are composed of proteins which in part play a role in resistance behaviour of spores to varied chemical

  14. 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.

  15. 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

    2015-11-05

    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.

  16. 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.

  17. Bacillus odysseyi isolate

    Science.gov (United States)

    Venkateswaran, Kasthuri (Inventor); La Duc, Myron Thomas (Inventor)

    2007-01-01

    The present invention relates to discovery and isolation of a biologically pure culture of a Bacillus odysseyi isolate with high adherence and sterilization resistant properties. B. odysseyi is a round spore forming Bacillus species that produces an exosporium. This novel species has been characterized on the basis of phenotypic traits, 16S rDNA sequence analysis and DNA-DNA hybridization. According to the results of these analyses, this strain belongs to the genus Bacillus and the type strain is 34hs-1.sup.T (=ATCC PTA-4993.sup.T=NRRL B-30641.sup.T=NBRC 100172.sup.T). The GenBank accession number for the 16S rDNA sequence of strain 34hs-1.sup.T is AF526913.

  18. Spore-Forming Bacteria that Resist Sterilization

    Science.gov (United States)

    LaDuc, Myron; Venkateswaran, Kasthuri

    2003-01-01

    A report presents a phenotypic and genotypic characterization of a bacterial species that has been found to be of the genus Bacillus and has been tentatively named B. odysseensis because it was isolated from surfaces of the Mars Odyssey spacecraft as part of continuing research on techniques for sterilizing spacecraft to prevent contamination of remote planets by terrestrial species. B. odysseensis is a Gram-positive, facultatively anaerobic, rod-shaped bacterium that forms round spores. The exosporium has been conjectured to play a role in the elevated resistance to sterilization. Research on the exosporium is proposed as a path toward improved means of sterilization, medical treatment, and prevention of biofouling.

  19. 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.

  20. Scanning surface potential microscopy of spore adhesion on surfaces.

    Science.gov (United States)

    Lee, I; Chung, E; Kweon, H; Yiacoumi, S; Tsouris, C

    2012-04-01

    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. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Contribution of endogenous and exogenous damage to the total radiation-induced damage in the bacterial spore

    International Nuclear Information System (INIS)

    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. 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.

  3. Monitoring of marine Bacillus diversity among the bacteria ...

    African Journals Online (AJOL)

    germination an indication of manganese reduction by Bacillus. Two strains identified as Bacillus pumilus using 16S rRNA gene sequence were isolated from Buzzers Bay seawater at 45 feet dept. The strains spores were centrally located and they were able to tolerate 1.0 M NaCl concentration indicating their marine origin.

  4. New pressure and temperature effects on bacterial spores

    International Nuclear Information System (INIS)

    Mathys, A; Knorr, D; Heinz, V

    2008-01-01

    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 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

  5. 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

  6. 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

  7. Dynamic phase microscopy, a new method to detect viable and killed spores and to estimate the heterogeneity of spore populations

    Science.gov (United States)

    Tychinsky, Vladimir P.; Mulyukin, Andrey L.; Lisovskii, Vitalii V.; Nikolaev, Yury A.; Kretushev, Aleksander V.; Vyshenskaya, Tatyana V.; Suzina, Nataliya E.; Duda, Vitalii I.; El-Registan, Galina I.

    One of the challenging tasks in monitoring studies is to estimate heterogeneity of microbial populations by the physiological state and potential viability of individual cells, especially with regard of their ability to withstand various environmental assaults. Previously, we described some approaches based on electron microscopy methods to discriminate vegetative, dormant, and dead cells in both aged microbial cultures and environmental samples, including permafrost. We propose to extend the arsenal of microscopy methods for monitoring studies by a new non-invasive and informative method - dynamic phase microscopy (DPM). The substantial advantage of DPM is that it gives quantitative (digitized) data of undestroyed (living) microscopic objects, exemplified in our work by Bacillus licheniformis spores. Using DPM made it possible to record interference images of objects (spores) and to produce picture of their "phase thickness" (PT) that is the optical path difference in nm. Thus, it was demonstrated the remarkable difference in the PT of spores at different physiological states: dormant, germinating, and heat-killed spores had PT values of 80, 40-50, and 20 nm, respectively. The other found criterion to distinguish between spores was the PT fluctuations. In contrast to dormant and killed spores, the PT of germinating spores oscillated with amplitude of up to 7 nm, with typical frequencies of 1.3 and 3.4 Hz. A combination of the recorded PT values and PT fluctuations gave a key to detect viable and dead cells. Under the conditions that did not support germination (the lack of nutrients), we were able to follow the response of a single dormant spore and a spore population to heating from 25 °C to 70 °C. Thus, a very small temperature change (from 40 °C to 42 °C) under conditions non-favorable for germination, caused a drastic decrease in the spores' PT; the second drop in the PT values was observed during heating from 60 °C to 70 °C. These changes were

  8. The contribution of endogenous and exogenous effects to radiation-induced damage in the bacterial spore

    International Nuclear Information System (INIS)

    Jacobs, G.P.; Samuni, A.; Czapski, G.

    1985-01-01

    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. (author)

  9. 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.

  10. Comparison of sampling methods to recover germinated Bacillus anthracis and Bacillus thuringiensis endospores from surface coupons.

    Science.gov (United States)

    Mott, T M; Shoe, J L; Hunter, M; Woodson, A M; Fritts, K A; Klimko, C P; Quirk, A V; Welkos, S L; Cote, C K

    2017-05-01

    In an attempt to devise decontamination methods that are both effective and minimally detrimental to the environment, we evaluated germination induction as an enhancement to strategies for Bacillus anthracis spore decontamination. To determine an optimal method for the recovery of germinating spores from different matrices, it was critical to ensure that the sampling procedures did not negatively impact the viability of the germinating spores possibly confounding the results and downstream analyses of field trial data. Therefore, the two main objectives of this study were the following: (i) development of an effective processing protocol capable of recovering the maximum number of viable germinating or germinated spores from different surface materials; and (ii) using a model system of spore contamination, employ this protocol to evaluate the potential applicability of germination induction to wide-area decontamination of B. anthracis spores. We examined parameters affecting the sampling efficiencies of B. anthracis and the surrogate species Bacillus thuringiensis on nonporous and porous materials. The most efficient extraction from all matrices was observed using PBS with 0·01% Tween 80 extraction buffer. The addition of a sonication and/or extended vortex treatment did not yield significant increases in spore or germinated spore recovery. Our data demonstrate that previous germination-induction experiments performed in suspension can be reproduced when Bacillus spores are deposited onto reference surfaces materials. Our proof of concept experiment illustrated that a germination pretreatment step significantly improves conventional secondary decontamination strategies and remediation plans. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  11. Inactivation of dried bacteria and bacterial spores by means of gamma irradiation at high temperatures.

    Science.gov (United States)

    Emborg, C

    1974-05-01

    Dried preparations with Streptococcus faecium, strain A(2)1, and spores of Bacillus sphaericus, strain C(I)A, normally used for control of the microbiological efficiency of radiation sterilization plants and preparations with spores of Bacillus subtilis, normally used for control of sterilization by dry heat, formalin, and ethylene oxide, as well as similar preparations with Micrococcus radiodurans, strain R(1), and spores of Bacillus globigii (B. subtilis, var. niger) were gamma irradiated with dose rates from 16 to 70 krad/h at temperatures from 60 to 100 C. At 80 C the radiation response of the spore preparations was the same as at room temperature, whereas the radiation resistance of the preparations with the two vegetative strains was reduced. At 100 C the radiation response of preparations with spores of B. subtilis was unaffected by the high temperature, whereas at 16 and and 25 krad/h the radiation resistance of the radiation-resistant sporeformer B. sphaericus, strain C(I)A, was reduced to the level of radiation resistance of preparations with spores of B. subtilis. It is concluded that combinations of heat and gamma irradiation at the temperatures and dose rates tested may have very few practical applications in sterilization of medical equipment.

  12. A mobile genetic element profoundly increases heat resistance of bacterial spores.

    Science.gov (United States)

    Berendsen, Erwin M; Boekhorst, Jos; Kuipers, Oscar P; Wells-Bennik, Marjon H J

    2016-11-01

    Bacterial endospores are among the most resilient forms of life on earth and are intrinsically resistant to extreme environments and antimicrobial treatments. Their resilience is explained by unique cellular structures formed by a complex developmental process often initiated in response to nutrient deprivation. Although the macromolecular structures of spores from different bacterial species are similar, their resistance to environmental insults differs widely. It is not known which of the factors attributed to spore resistance confer very high-level heat resistance. Here, we provide conclusive evidence that in Bacillus subtilis, this is due to the presence of a mobile genetic element (Tn1546-like) carrying five predicted operons, one of which contains genes that encode homologs of SpoVAC, SpoVAD and SpoVAEb and four other genes encoding proteins with unknown functions. This operon, named spoVA 2mob , confers high-level heat resistance to spores. Deletion of spoVA 2mob in a B. subtilis strain carrying Tn1546 renders heat-sensitive spores while transfer of spoVA 2mob into B. subtilis 168 yields highly heat-resistant spores. On the basis of the genetic conservation of different spoVA operons among spore-forming species of Bacillaceae, we propose an evolutionary scenario for the emergence of extremely heat-resistant spores in B. subtilis, B. licheniformis and B. amyloliquefaciens. This discovery opens up avenues for improved detection and control of spore-forming bacteria able to produce highly heat-resistant spores.

  13. Sensitivity of Dormant and Germinating B, Anthracis Spores to Polycationic Compound

    Science.gov (United States)

    2005-06-01

    practical problems such as food spoilage , degradation of industrial materials, and "sick building syndrome" resulting from colonization of ventilation systems...species of Clostridium and Bacillus are of concern to the food industry due to their potential for spoilage of or toxin production in improperly...1.2. While most spore-forming bacterial species are classified as non-pathogenic or opportunistically pathogenic, Bacillus anthracis is well-known a

  14. 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.

  15. Microrganismos heterotróficos mesófilos e bactérias do grupo do Bacillus cereus em leite integral submetido a ultra alta temperatura Mesophilic heterotrophic microorganisms and spore forming bacteria from Bacillus cereus group in ultra high temperature milk

    Directory of Open Access Journals (Sweden)

    A.M.C. Vidal-Martins

    2005-06-01

    Full Text Available Cento e dez amostras de 11 diferentes marcas de leite ultra alta temperatura (UAT, comercializadas em São José do Rio Preto - SP, foram submetidas à contagem de microrganismos heterotróficos mesófilos viáveis e à pesquisa de bactérias do grupo do Bacillus cereus. A população de microrganismos mesófilos variou de ´10² UFC/ml a >1,0´10(6 UFC/ml. Bactérias do grupo do Bacillus cereus foram verificadas em 13 (11,8% amostras. Os resultados evidenciaram elevada população de microrganismos indicadores mesófilos.One hundred and ten ultra high temperature (UHT milk samples, from 11 different brands retailed in São José do Rio Preto - SP were analysed for mesophilic heterotrophic microorganisms counting, as well as for the presence of Bacillus cereus. Mesophilic heterotrophic microorganisms ranged from ´10² CFU/ml to >1.0´10(6 CFU/ml. Bacillus cereus was detected in 13 (11.8% samples. The results demonstrated high countings of mesophilic microorganisms.

  16. Germination and persistence of Bacillus anthracis and Bacillus thuringiensis in soil microcosms.

    Science.gov (United States)

    Bishop, A H

    2014-11-01

    Decontaminating large, outdoor spaces of Bacillus anthracis spores presents significant problems, particularly in soil. Proof was sought that the addition of germinant chemicals could cause spores of B. anthracis and Bacillus thuringiensis, a commonly used simulant of the threat agent, to convert to the less resistant vegetative form in a microcosm. Nonsterile plant/soil microcosms were inoculated with spores of B. thuringiensis and two nonpathogenic strains of B. anthracis. A combination of L-alanine (100 mmol l(-1)) and inosine (10 mmol l(-1)) resulted in a 6 log decrease in spore numbers in both strains of B. anthracis over 2 weeks at 22°C; a 3 log decrease in B. anthracis Sterne spore numbers was observed after incubation for 2 weeks at 10°C. Negligible germination nor a decrease in viable count occurred in either strain when the concentration of L-alanine was decreased to 5 mmol l(-1). Germinated spores of B. thuringiensis were able to persist in vegetative form in the microcosms, whereas those of B. anthracis rapidly disappeared. The pleiotropic regulator PlcR, which B. anthracis lacks, does not contribute to the persistence of B. thuringiensis in vegetative form in soil. The principle of adding germinants to soil to trigger the conversion of spores to vegetative form has been demonstrated. Bacillus anthracis failed to persist in vegetative form or resporulate in the microcosms after it had been induced to germinate. The large scale, outdoor decontamination of B. anthracis spores may be facilitated by the application of simple, defined combinations of germinants. © 2014 Crown Copyright. Journal of Applied Microbiology © 2014 Society for Applied Microbiology This article is Published with the permission of the Controller of HMSO and Queen's Printer for Scotland.

  17. Longitudinal assessment of dairy farm management practices associated with the presence of psychrotolerant Bacillales spores in bulk tank milk on 10 New York State dairy farms

    NARCIS (Netherlands)

    Masiello, S. N.; Kent, D.V.; Martin, N. H.; Schukken, Y. H.; Wiedmann, M.; Boor, K. J.

    2017-01-01

    The ability of certain spore-forming bacteria in the order Bacillales (e.g., Bacillus spp., Paenibacillus spp.) to survive pasteurization in spore form and grow at refrigeration temperatures results in product spoilage and limits the shelf life of high temperature, short time (HTST)-pasteurized

  18. 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.

  19. Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

    International Nuclear Information System (INIS)

    Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I.

    1990-01-01

    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

  20. Investigation of spore forming bacterial flooding for enhanced oil recovery in a North Sea chalk Reservoir

    DEFF Research Database (Denmark)

    Halim, Amalia Yunita; Nielsen, Sidsel Marie; Eliasson Lantz, Anna

    2015-01-01

    Bacillus licheniformis 421 was used as it was shown to be a good candidate in a previous study. Bacterial spore can penetrate deeper into the chalk rock, squeezing through the pore throats. Our results showed that injection of B. licheniformis 421 as a tertiary oil recovery method, in the residual oil...

  1. Roles of DacB and spm proteins in clostridium perfringens spore resistance to moist heat, chemicals, and UV radiation.

    Science.gov (United States)

    Paredes-Sabja, Daniel; Sarker, Nahid; Setlow, Barbara; Setlow, Peter; Sarker, Mahfuzur R

    2008-06-01

    Clostridium perfringens food poisoning is caused mainly by enterotoxigenic type A isolates that typically possess high spore heat resistance. Previous studies have shown that alpha/beta-type small, acid-soluble proteins (SASP) play a major role in the resistance of Bacillus subtilis and C. perfringens spores to moist heat, UV radiation, and some chemicals. Additional major factors in B. subtilis spore resistance are the spore's core water content and cortex peptidoglycan (PG) structure, with the latter properties modulated by the spm and dacB gene products and the sporulation temperature. In the current work, we have shown that the spm and dacB genes are expressed only during C. perfringens sporulation and have examined the effects of spm and dacB mutations and sporulation temperature on spore core water content and spore resistance to moist heat, UV radiation, and a number of chemicals. The results of these analyses indicate that for C. perfringens SM101 (i) core water content and, probably, cortex PG structure have little if any role in spore resistance to UV and formaldehyde, presumably because these spores' DNA is saturated with alpha/beta-type SASP; (ii) spore resistance to moist heat and nitrous acid is determined to a large extent by core water content and, probably, cortex structure; (iii) core water content and cortex PG cross-linking play little or no role in spore resistance to hydrogen peroxide; (iv) spore core water content decreases with higher sporulation temperatures, resulting in spores that are more resistant to moist heat; and (v) factors in addition to SpmAB, DacB, and sporulation temperature play roles in determining spore core water content and thus, spore resistance to moist heat.

  2. Induced Sporicidal Activity of Chlorhexidine against Clostridium difficile Spores under Altered Physical and Chemical Conditions

    Science.gov (United States)

    Nerandzic, Michelle M.; Donskey, Curtis J.

    2015-01-01

    Background Chlorhexidine is a broad-spectrum antimicrobial commonly used to disinfect the skin of patients to reduce the risk of healthcare-associated infections. Because chlorhexidine is not sporicidal, it is not anticipated that it would have an impact on skin contamination with Clostridium difficile, the most important cause of healthcare-associated diarrhea. However, although chlorhexidine is not sporicidal as it is used in healthcare settings, it has been reported to kill spores of Bacillus species under altered physical and chemical conditions that disrupt the spore’s protective barriers (e.g., heat, ultrasonication, alcohol, or elevated pH). Here, we tested the hypothesis that similarly altered physical and chemical conditions result in enhanced sporicidal activity of chlorhexidine against C. difficile spores. Principal Findings C. difficile spores became susceptible to heat killing at 80°C within 15 minutes in the presence of chlorhexidine, as opposed to spores suspended in water which remained viable. The extent to which the spores were reduced was directly proportional to the concentration of chlorhexidine in solution, with no viable spores recovered after 15 minutes of incubation in 0.04%–0.0004% w/v chlorhexidine solutions at 80°C. Reduction of spores exposed to 4% w/v chlorhexidine solutions at moderate temperatures (37°C and 55°C) was enhanced by the presence of 70% ethanol. However, complete elimination of spores was not achieved until 3 hours of incubation at 55°C. Elevating the pH to ≥9.5 significantly enhanced the killing of spores in either aqueous or alcoholic chlorhexidine solutions. Conclusions Physical and chemical conditions that alter the protective barriers of C. difficile spores convey sporicidal activity to chlorhexidine. Further studies are necessary to identify additional agents that may allow chlorhexidine to reach its target within the spore. PMID:25861057

  3. Population and function analysis of cultivable bacteria associated with spores of arbuscular mycorrhizal fungus Gigaspora margarita.

    Science.gov (United States)

    Long, Liangkun; Lin, Qunying; Yao, Qing; Zhu, Honghui

    2017-05-01

    This study was aimed to investigate the diversity and function of bacterial population associated with Gigaspora margarita spores. The fungus was propagated in sterilized sand/soil pots using alfalfa (Medicago sativa), grain sorghum (Sorghum bicolor), or maize (Zea mays) as host plants, or in sterilized vermiculite pots using alfalfa as host plants, respectively. Bacteria were isolated from the new-formed spores using diluted plate method, and typical bacterial isolates were identified according to 16S rRNA gene phylogenetic analysis. Total 43 bacterial isolates affiliated to three phyla and 23 genera were obtained. The spore-associated bacterial communities were obviously different among the four source spores, suggesting that plant species or substrates could influence the bacterial population. Bacillus and Streptomyces were most frequently associated with the fungal spores. Function analysis of these bacteria by plate tests, it was found that about 30.2% isolates stimulated the spore germination, five out of seven tested isolates improved the hyphal growth, total 57.5% of the tested isolates solubilized phosphorus at different levels, 15% isolates degraded chitin, and a few isolates suppressed the growth of Escherichia coli or Staphylococcus aureus. In pot experiment, three bacterial isolates (belonging to Curtobacterium, Ensifer, or Bacillus, respectively) displayed improvement effect on alfalfa growth and/or the colonization of roots by G. margarita.

  4. DNA capturing machinery through spore-displayed proteins.

    Science.gov (United States)

    Park, T J; Lee, S J; Pan, J-G; Jung, H-C; Park, J Y; Park, J P; Lee, S Y

    2011-10-01

    The purpose of this study was to develop a general method for the facile development of a new DNA biosensor which utilizes streptavidin-displayed spores as a molecular machinery. Fluorescence spectroscopy was used as a monitoring tool for the streptavidin displayed on the surface of Bacillus thuringiensis spores and as a diagnosis method for DNA detection. As a proof-of-concept, four pathogenic bacteria including Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumonia were used for the detection of pathogenic species. In addition, a set of mutant variants of Wilson's disease were also used for the detection of single nucleotide polymorphism (SNP) in this system. This strategy, utilizing streptavidin-displayed spores, is capable of capturing DNA targets for the detection of pathogenic bacteria and for mutation analysis in Wilson's disease. This approach could be useful as a simple platform for developing sensitive spore-based biosensors for any desired DNA targets in diagnostic applications. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

  5. Influence of Bacillus polymyxa on the growth and development of Fusarium oxysporum f. sp. tulipae

    Directory of Open Access Journals (Sweden)

    Alicja Saniewska

    2013-12-01

    Full Text Available Antagonistic effect of Bacillus polymyxa, strain S13, toward Fusarium oxysporum f. sp. tulipae was evaluated iii vitro and in vivo. The growth of the pathogen was greatly inhibited in dual cultures with Bacillus polymyxa on potato dextrose agar. Suspension of B. polymyxa and its filtrate substantially inhibited spore germination and development of Fusarium oxysporuum f. sp. tulipae on tulip bulbs.

  6. Inhibition of Lipopolysaccharide-Induced Interleukin 8 in Human Adenocarcinoma Cell Line HT-29 by Spore Probiotics: B. coagulans and B. subtilis (natto).

    Science.gov (United States)

    Azimirad, Masoumeh; Alebouyeh, Masoud; Naji, Tahereh

    2017-03-01

    Probiotics are used as a treatment for different intestinal disorders. They confer health benefits by different ways. This study was aimed to investigate immunomodulatory effect of Bacillus probiotic spores on the production of lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) in HT-29 intestinal epithelial cells. Differentiated intestinal epithelial cell line was used as a model for the study of colonization of purified spores (Bacillus subtilis (natto) and B. coagulans) and their anti-inflammatory effects. MTT assay and trypan blue staining were used for the detection of optimal concentration of the purified spores and LPS. Pre-treatment assay was done by treatment of the cells with the purified spores for 2 h, followed by challenges with LPS for 3 and 18 h. Post-treatment assay was done by initial treatment of the cells with LPS for 18 h, followed by the spores for 3 and 6 h. Levels of IL-8 secretion and its mRNA expression were measured by ELISA and relative Q real-time PCR. Our results showed similar rates of adherence to intestinal epithelial cells by the spore probiotics, while displaying no cytotoxic effect. In the pre-treatment assay, a significant decrease in IL-8, at both protein and mRNA levels, was measured for B. coagulans spores after the addition of LPS, which was higher than those observed for Bacillus subtilis (natto) spores. In the post-treatment assay, while Bacillus subtilis (but not B. coagulans) diminished the LPS-stimulated IL-8 levels after 3 h of incubation, the inhibitory effect was not constant. In conclusion, ability of Bacillus spore probiotics for adherence to intestinal epithelial cell and their anti-inflammatory effects, through interference with LPS/IL-8 signaling, was shown in this study. Further studies are needed to characterize responsible bacterial compounds associated with these effects.

  7. [Analysis of bacterial colonization associated with Gigaspora margarita spores by green fluorescence protein (GFP) marked technology].

    Science.gov (United States)

    Long, Liangkun; Yao, Qing; Ai, Yuncan; Zhu, Honghui

    2009-05-01

    We analyzed bacterial colonization associated with spores of arbuscular mycorrhizal fungi (AMF) Gigaspora margarita, to indicate their ecological niche, and to provide information for further researches on their populations or functions. Six bacteria strains (Peanibacillus sp. M060106-1, Peanibacillus sp. M061122-2, Peanibacillus sp. M061122-6, Bacillus sp. M061122-4, Bacillus sp. M061122-10 and Brevibacillus sp. M061122-12) isolated from G. margarita spores were tagged with green fluorescence protein (GFP) using the carrier plasmid pNF8 (gfp-mut1). We analyzed the ecological niche and population dynamics of tagged strains on G. margarita under different conditions by using fluorescent microscope and/or plate counts. Four strains (M060106-1, M061122-6, M061122-10 and M061122-12) were tagged with GFP, showing high plasmid stability. These tagged strains possessed the basic characteristics identical to their original strains and, hence, were fit for short-term study of environmental colonization. All four GFP-tagged strains colonized the spore wall of G. margarita, and M061122-6 and M061122-12 further colonized the fungal hyphae. Under different pH conditions,the population dynamic of each GFP-tagged strain on the spores showed the same trend, i.e. first increased and then decreased, and the effects on the population size varied with different pH value. GFP-tagged strains colonized the spores of low viability more easily than those of high viability, and the population dynamic on the spores of high viability was different for each tagged strain. The isolated bacteria associated with G. margarita spores can re-colonize the fungal spores, whereas their colonizing ability depends on their characteristics and environmental factors. These data contributes to the further understanding of populations and functions of AMF-associated bacteria.

  8. Computational Fluid Dynamics Modeling of Bacillus anthracis ...

    Science.gov (United States)

    Journal Article 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. Four 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. Despite the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the upper conducting airways of the human at the same air concentration of anthrax spores. This greater deposition of spores in the upper airways in the human resulted in lower penetration and deposition in the tracheobronchial airways and the deep lung than that predict

  9. Complete Genomes of Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, Two Phylogenetically Distinct Probiotics

    Science.gov (United States)

    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. PMID:27258038

  10. 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.

  11. 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.

  12. Sensitivity of the bacterium Bacillus Thuringiensis as an insect disease agent to gamma-rays

    International Nuclear Information System (INIS)

    Merdam, A.I.; Abdu, R.M.

    1977-01-01

    The effect of gamma radiation on the viability of the entomopathogenic spore-forming bacterium, Bacillus thuringiensis, was tested. The different gamma doses varied much in their effect on such bacterium. All irradiated Bacillus suspensions with doses below 85 krad showed different degrees of inhibitory activity. However, bacterial suspensions irradiated at a dose of 90 krad. proved to promote spore germination. Changes in the physiological, and morphological characters of the irradiated Bacillus at these levels were detected. The new observed characters were induced at a particular dose level of 90 krad. These new characters are assumed to be due to genetic changes induced at this particular gamma dose

  13. 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.

  14. Development of method for evaluating cell hardness and correlation between bacterial spore hardness and durability

    Science.gov (United States)

    2012-01-01

    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. PMID:22676476

  15. 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.

  16. Role of germinant receptors in Caco-2 cell-initiated germination of Bacillus cereus ATCC 14579 endospores

    NARCIS (Netherlands)

    Hornstra, L.M.; Voort, van der M.; Wijnands, L.M.; Roubos-van den Hil, P.J.; Abee, T.

    2009-01-01

    Spores obtained from Bacillus cereus ATCC 14579 and mutant strains lacking each of seven germinant receptor operons were exposed to differentiated Caco-2 cells and monitored for germination. Spores of the gerI and gerL mutants showed a reduced germination response, pointing to a role for these

  17. Bacillus velezensis sp. nov., a surfactant-producing bacterium isolated from the river Vélez in Málaga, southern Spain.

    Science.gov (United States)

    Ruiz-García, Cristina; Béjar, Victoria; Martínez-Checa, Fernando; Llamas, Inmaculada; Quesada, Emilia

    2005-01-01

    Two Gram-positive, endospore-forming bacterial strains, CR-502T and CR-14b, which produce surfactant molecules are described. Phenotypic tests and phylogenetic analyses showed these strains to be members of the genus Bacillus and related to the species Bacillus atrophaeus, Bacillus mojavensis, Bacillus subtilis, Bacillus vallismortis and Bacillus amyloliquefaciens, although they differ from these species in a number of phenotypic characteristics. DNA-DNA hybridization confirmed that they show less than 20 % hybridization with the above-mentioned species and therefore represent a novel species of Bacillus. The DNA G+C content is 46.4 mol% in strain CR-502T and 46.1 mol% in strain CR-14b. The main fatty acids in strain CR-502T are 15 : 0 anteiso (32.70 %), 15 : 0 iso (29.86 %) and 16 : 0 (13.41 %). The main quinone in strain CR-502T is MK-7 (96.6 %). In the light of the polyphasic evidence gathered in this study, it is proposed that these strains be classified as a novel species of the genus Bacillus, with the name Bacillus velezensis sp. nov. The type strain (CR-502T=CECT 5686T=LMG 22478T) was isolated from a brackish water sample taken from the river Vélez at Torredelmar in Málaga, southern Spain.

  18. Amplicon sequencing for the quantification of spoilage microbiota in complex foods including bacterial spores.

    Science.gov (United States)

    de Boer, Paulo; Caspers, Martien; Sanders, Jan-Willem; Kemperman, Robèr; Wijman, Janneke; Lommerse, Gijs; Roeselers, Guus; Montijn, Roy; Abee, Tjakko; Kort, Remco

    2015-01-01

    Spoilage of food products is frequently caused by bacterial spores and lactic acid bacteria. Identification of these organisms by classic cultivation methods is limited by their ability to form colonies on nutrient agar plates. In this study, we adapted and optimized 16S rRNA amplicon sequencing for quantification of bacterial spores in a canned food matrix and for monitoring the outgrowth of spoilage microbiota in a ready-to-eat food matrix. The detection limit of bar-coded 16S rRNA amplicon sequencing was determined for the number of bacterial spores in a canned food matrix. Analysis of samples from a canned food matrix spiked with a mixture of equinumerous spores from the thermophiles, Geobacillus stearothermophilus and Geobacillus thermoglucosidans, and the mesophiles, Bacillus sporothermodurans, Bacillus cereus, and Bacillus subtilis, led to the detection of these spores with an average limit of 2 × 10(2) spores ml(-1). The data were normalized by setting the number of sequences resulting from DNA of an inactivated bacterial species, present in the matrix at the same concentration in all samples, to a fixed value for quantitative sample-to-sample comparisons. The 16S rRNA amplicon sequencing method was also employed to monitor population dynamics in a ready-to-eat rice meal, incubated over a period of 12 days at 7 °C. The most predominant outgrowth was observed by the genera Leuconostoc, Bacillus, and Paenibacillus. Analysis of meals pre-treated with weak acids showed inhibition of outgrowth of these three genera. The specificity of the amplicon synthesis was improved by the design of oligonucleotides that minimize the amplification of 16S rRNA genes from chloroplasts originating from plant-based material present in the food. This study shows that the composition of complex spoilage populations, including bacterial spores, can be monitored in complex food matrices by bar-coded amplicon sequencing in a quantitative manner. In order to allow sample

  19. Enhanced biomass production study on probiotic Bacillus subtilis ...

    African Journals Online (AJOL)

    The culture conditions of lactose fermenting, spore forming probiotic Bacillus subtilis SK09 isolated from dairy effluent were optimized by response surface methodology to maximize the biomass production. The student's t-test of the Placket-Burman screening design revealed that the effects of pH, ammonium citrate and ...

  20. Virulence of Bacillus cereus as natural facultative pathogen of ...

    African Journals Online (AJOL)

    Out of 4407 Anopheles subpictus larvae collected from submerged rice-fields and shallow ponds, 1412 were found to be unhealthy and 2.8% of unhealthy larvae were naturally infected by Bacillus cereus. B. cereus formed circular, white and flat colonies. Bacteria were gram positive, ellipsoidal/oval spore forming aerobic ...

  1. Enhanced biomass production study on probiotic Bacillus subtilis ...

    African Journals Online (AJOL)

    user

    2010-11-22

    Nov 22, 2010 ... The culture conditions of lactose fermenting, spore forming probiotic Bacillus subtilis SK09 isolated from dairy effluent were optimized by response surface methodology to maximize the biomass production. The student's t-test of the Placket-Burman screening design revealed that the effects of pH,.

  2. Effect of oral administration of Bacillus coagulans B37 and Bacillus pumilus B9 strains on fecal coliforms, Lactobacillus and Bacillus spp. in rat animal model.

    Science.gov (United States)

    Haldar, Lopamudra; Gandhi, D N

    2016-07-01

    To investigate the effect of oral administration of two Bacillus strains on fecal coliforms, Lactobacillus and Bacillus spp. in rat animal model. An in vivo experiment was conducted for 49-day period on 36 adult male albino Wister rats divided equally into to four groups. After 7-day adaptation period, one group (T1) was fed on sterile skim milk along with basal diet for the next 28 days. Second (T2) and (T3) groups received spore biomass of Bacillus coagulans B37 and Bacillus pumilus B9, respectively, suspended in sterilized skim milk at 8-9 log colony-forming units/ml plus basal diet for 28 days, while control group (T4) was supplied with clean water along with basal diet. There was a 14-day post-treatment period. A total of 288 fecal samples (8 fecal collections per rat) were collected at every 7-day interval starting from 0 to 49 days and subjected to the enumeration of the counts of coliforms and lactobacilli and Bacillus spores using respective agar media. In vitro acid and bile tolerance tests on both the strains were performed. The rats those (T2 and T3) received either B. coagulans B37 or B. pumilus B9 spore along with non-fermented skim milk showed decrease (pBacillus spore counts as compared to the control group (T4) and the group fed only skim milk (T1). In vitro study indicated that both the strains were found to survive at pH 2.0 and 3.0 even up to 3 h and tolerate bile up to 2.0% concentration even after 12 h of exposure. This study revealed that oral administration of either B. coagulans B37 or B. pumilus B9 strains might be useful in reducing coliform counts accompanied by concurrent increase in lactobacilli counts in the intestinal flora in rats.

  3. Bacillus cucumis

    Science.gov (United States)

    Kämpfer, Peter; Busse, Hans-Jürgen; Glaeser, Stefanie P; Kloepper, Joseph W; Hu, Chia-Hui; McInroy, John A

    2016-02-01

    A facultative anaerobic, Gram-positive staining, endospore-forming bacterium, isolated from the rhizosphere of cucumber ( Cucumis sativus ), was taxonomically investigated. Based on 16S rRNA gene sequence similarity comparisons, strain AP-6 T clustered together with other species of the genus Bacillus and showed highest similarities with Bacillus drentensis LMG 21831 T (99.1 %), Bacillus vireti LMG 21834 T (98.7 %) and Bacillus soli LMG 21838 T (98.5 %). The 16S rRNA gene sequence similarity to the sequences of the type strains of other species of the genus Bacillus was 98.5 % or less. Chemotaxonomic features supported the grouping of the strain in the genus Bacillus ; for example, the major fatty acids were anteiso-C 15 : 0 , iso-C 15 : 0 and C 16 : 0 , the polar lipid profile contained the major components diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified glycolipid, the major quinone was menaquinone MK-7 and the major compound in the polyamine pattern was spermidine. Additionally, DNA-DNA hybridization with B. drentensis LMG 21831 T , B. vireti LMG 21834 T and B. soli LMG 21838 T resulted in relatedness values that were clearly below 70 %. Physiological and biochemical test results were also different from those of the most closely related species. As a consequence, AP-6 T represents a novel species of the genus Bacillus , for which the name Bacillus cucumis sp. nov. is proposed, with AP-6 T ( = CIP 110974 T  = CCM 8651 T ) as the type strain.

  4. Thermal Inactivation of Bacillus anthracis Spores Using Rapid Resistive Heating

    Science.gov (United States)

    2016-03-24

    persist in the environment over millennial time spans in a metabolically inactive state (Nicholson et al., 2000)." Once favorable conditions arise...for the prototyping/initial testing, the collection of 1586 data points, and to ensure quality agar plates were used for the thermal inactivation...removal process yielded some broken filament samples and required inspection for cracks of still intact filament samples to ensure quality samples

  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 8×107 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 (PBacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological 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. PMID:23029244

  6. 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 (PBacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological 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.

  7. The Survival and Recovery of Irradiated Bacterial Spores as Affected by Population Density and Some External Factors

    International Nuclear Information System (INIS)

    Farkas, J.; Kiss, I.; Andrássy, E.

    1967-01-01

    The radiation resistance of Bacillus cereus spores as affected by the pH-value and cell density of the irradiated spore suspensions was investigated. The portions of the survival curves of suspensions of 10 8 , 4 x 10 3 and 5 x 10 1 per millilitre viable cell counts, respectively, were compared for a three-orders-of-magnitude decrease in viable cell count. It was established that the initial cell density did not affect radiation resistance of spores. Radiation resistance as affected by pH-value in the range of 3 to 8 was investigated. In the range of pH 5 to 8, the radiation resistance of B. cereus spores was not affected. By lowering the pH-value to below 5, the radiation resistance decreased below that observed in the neutral region. The colony-forming capacity of B. cereus, B. coagulans and B. pumilus as a function of the pH-value in the nutrient medium, and the pH-sensitivity of bacterial spores as affected by radiation, were also investigated. It was established that irradiation increased the pH-sensitivity of surviving bacterial spores in all three strains. The initial phase of spore germination (the phase accompanied by decrease of refractivity of the spores) and the division stage of vegetative cells proved to be the most sensitive to the value of the hydrogen ion concentration. (author)

  8. Proposal of a simplified technique for staining bacterial spores without applying heat--successful modification of Moeller's method.

    Science.gov (United States)

    Hayama, M; Oana, K; Kozakai, T; Umeda, S; Fujimoto, J; Ota, H; Kawakami, Y

    2007-08-16

    As the bacterial spores are difficult to stain, a number of staining techniques including their modifications have been proposed to date. Most of the conventional staining procedures unexceptionally contain the step of staining with steamed dye reagent in order to increase the stainability of the spores. We made an attempt to improve the conventional Moeller's methods for staining bacterial spores. Spores of Bacillus species were stained with our modified Moeller's spore stain and evaluated for its staining properties. We investigated the stainability of both of the conventional and the modified Moeller's methods and the evaluation was made whether or not the step of steaming of Kinyoun's carbol-fuchsine dye reagent could be omitted by adding to aliquots of Tergitol 7, in place of the conventional dye solution steamed for some interval over hot blue flame of a Bunsen burner. We successfully omitted the heating step of steaming the Kinyoun's carbol fuchsine dye solution in the Moeller's method of bacterial spore stain, by the replacement of Kinyoun's carbol-fuchsine dye solution involving 2 drops of Tergitol 7, nonionic polyglycol ether surfactants type NP-7 (Sigma-Aldrich Japan, Tokyo, Japan) per 10 ml of Kinyoun's carbol-fuchsine dye solution. Bacillus spores stained pink to red and vegetative bacterial cells stained blue, although without applying any heating step during the whole course of staining processes including the fixation process. The novel staining method of our proposal resulted in far better satisfactory stainability in comparison with the conventional Moeller's method with the steaming dye solution. The modified spore stain without applying any heating step using the Kinyoun's carbol-fuchsine dye solution with an addition of Tergitol 7 aliquots was demonstrated to be reproducible and yielded consistent and satisfactory stainability. This simplified staining procedure is rapid to perform and found to be applicable to detect the bacterial spores in

  9. Deposition of Bacteria and Bacterial Spores by Bathroom Hot Air Hand Dryers.

    Science.gov (United States)

    Del Carmen Huesca-Espitia, Luz; Aslanzadeh, Jaber; Feinn, Richard; Joseph, Gabrielle; Murray, Thomas S; Setlow, Peter

    2018-02-09

    Hot air hand dryers in multiple men's and women's bathrooms in 3 basic science research areas in an academic health center were screened for their deposition on plates of: i) total bacteria, some of which were identified; and ii) a kanamycin resistant Bacillus subtilis strain, PS533, spores of which are produced in large amounts in one basic science research laboratory. Plates exposed to hand dryer air for 30 seconds averaged 18-60 colonies/plate but interior hand dryer nozzle surfaces had minimal bacterial levels, plates exposed to bathroom air for 2 minutes with hand dryers off averaged ≤1 colony, and plates exposed to bathroom air moved by a small fan for 20 minutes had averages of 15 and 12 colonies/plate in two buildings tested. Retrofitting hand dryers with HEPA filters reduced bacterial deposition by hand dryers ∼4-fold, and potential human pathogens were recovered from plates exposed to hand dryer air whether or not a HEPA filter was present, and from bathroom air moved by a small fan. Spore-forming colonies, identified as B. subtilis PS533 averaged ∼2.5-5% of bacteria deposited by hand dryers throughout basic research areas examined regardless of distance from the spore forming laboratory, and these were almost certainly deposited as spores. Comparable results were obtained when bathroom air was sampled for spores. These results indicate that many kinds of bacteria, including potential pathogens and spores, can be deposited on hands exposed to bathroom hand dryers, and that spores could be dispersed throughout buildings and deposited on hands by hand dryers. Importance While there is evidence that bathroom hand dryers can disperse bacteria from hands or deposit bacteria on surfaces, including recently washed hands, there is less information on: i) the organisms dispersed by hand dryers; ii) if hand dryers provide a reservoir of bacteria or simply blow large amounts of bacterially contaminated air; and iii) if bacterial spores are deposited on

  10. Spores of Clostridium difficile clinical isolates display a diverse germination response to bile salts.

    Directory of Open Access Journals (Sweden)

    Daniela Heeg

    Full Text Available Clostridium difficile spores play a pivotal role in the transmission of infectious diarrhoea, but in order to cause disease spores must complete germination and return to vegetative cell growth. While the mechanisms of spore germination are well understood in Bacillus, knowledge of C. difficile germination remains limited. Previous studies have shown that bile salts and amino acids play an important role in regulating the germination response of C. difficile spores. Taurocholate, in combination with glycine, can stimulate germination, whereas chenodeoxycholate has been shown to inhibit spore germination in a C. difficile clinical isolate. Our recent studies of C. difficile sporulation characteristics have since pointed to substantial diversity among different clinical isolates. Consequently, in this study we investigated how the germination characteristics of different C. difficile isolates vary in response to bile salts. By analysing 29 isolates, including 16 belonging to the BI/NAP1/027 type, we show that considerable diversity exists in both the rate and extent of C. difficile germination in response to rich medium containing both taurocholate and glycine. Strikingly, we also show that although a potent inhibitor of germination for some isolates, chenodeoxycholate does not inhibit the germination, or outgrowth, of all C. difficile strains. Finally, we provide evidence that components of rich media may induce the germination of C. difficile spores, even in the absence of taurocholate. Taken together, these data suggest that the mechanisms of C. difficile spore germination in response to bile salts are complex and require further study. Furthermore, we stress the importance of studying multiple isolates in the future when analysing the nutrients or chemicals that either stimulate or inhibit C. difficile spore germination.

  11. Isolation and genetic identification of spore-forming bacteria associated with concentrated-milk processing in Nebraska.

    Science.gov (United States)

    Martinez, Bismarck A; Stratton, Jayne; Bianchini, Andreia

    2017-02-01

    Spore-forming bacteria are heat-resistant microorganisms capable of surviving and germinating in milk after pasteurization. They have been reported to affect the quality of dairy products by the production of enzymes (lipolytic and proteolytic) under low-temperature conditions in fluid milk, and have become a limiting factor for milk powder in reaching some selective markets. The objective of this research was to isolate and identify the population of spore-forming bacteria (psychrotrophic and thermophilic strains) associated with concentrated milk processing in Nebraska. During 2 seasons, in-process milk samples from a commercial plant (raw, pasteurized, and concentrated) were collected and heat-treated (80°C/12 min) to recover only spore-formers. Samples were spread-plated using standard methods agar and incubated at 32°C to enumerate mesophilic spore counts. Heat-treated samples were also stored at 7°C and 55°C to recover spore-formers that had the ability to grow under those temperature conditions. Isolates obtained from incubation or storage conditions were identified using molecular techniques (16S or rpoB sequencing). Based on the identification of the isolates and their relatedness, strains found in raw, pasteurized, and concentrated milk were determined to be similar. Paenibacillus spp. were associated with both raw and concentrated milk. Due to their known ability to cause spoilage under refrigeration, this shows the potential risk associated with the transferring of these problematic organisms into other dairy products. Other Bacillus species found in concentrated milk included Bacillus clausii, Bacillus subtilis, Lysinibacillus sp., Bacillus safensis, Bacillus licheniformis, Bacillus sonorensis, and Brevibacillus sp., with the last 3 organisms being capable of growing at thermophilic temperatures. These strains can also be translocated to other dairy products, such as milk powder, representing a quality problem. The results of this research

  12. 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...... 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...... 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...

  13. Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis.

    Science.gov (United States)

    Han, Cliff S; Xie, Gary; Challacombe, Jean F; Altherr, Michael R; Bhotika, Smriti S; Brown, Nancy; Bruce, David; Campbell, Connie S; Campbell, Mary L; Chen, Jin; Chertkov, Olga; Cleland, Cathy; Dimitrijevic, Mira; Doggett, Norman A; Fawcett, John J; Glavina, Tijana; Goodwin, Lynne A; Green, Lance D; Hill, Karen K; Hitchcock, Penny; Jackson, Paul J; Keim, Paul; Kewalramani, Avinash Ramesh; Longmire, Jon; Lucas, Susan; Malfatti, Stephanie; McMurry, Kim; Meincke, Linda J; Misra, Monica; Moseman, Bernice L; Mundt, Mark; Munk, A Christine; Okinaka, Richard T; Parson-Quintana, B; Reilly, Lee Philip; Richardson, Paul; Robinson, Donna L; Rubin, Eddy; Saunders, Elizabeth; Tapia, Roxanne; Tesmer, Judith G; Thayer, Nina; Thompson, Linda S; Tice, Hope; Ticknor, Lawrence O; Wills, Patti L; Brettin, Thomas S; Gilna, Paul

    2006-05-01

    Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are closely related gram-positive, spore-forming bacteria of the B. cereus sensu lato group. While independently derived strains of B. anthracis reveal conspicuous sequence homogeneity, environmental isolates of B. cereus and B. thuringiensis exhibit extensive genetic diversity. Here we report the sequencing and comparative analysis of the genomes of two members of the B. cereus group, B. thuringiensis 97-27 subsp. konkukian serotype H34, isolated from a necrotic human wound, and B. cereus E33L, which was isolated from a swab of a zebra carcass in Namibia. These two strains, when analyzed by amplified fragment length polymorphism within a collection of over 300 of B. cereus, B. thuringiensis, and B. anthracis isolates, appear closely related to B. anthracis. The B. cereus E33L isolate appears to be the nearest relative to B. anthracis identified thus far. Whole-genome sequencing of B. thuringiensis 97-27and B. cereus E33L was undertaken to identify shared and unique genes among these isolates in comparison to the genomes of pathogenic strains B. anthracis Ames and B. cereus G9241 and nonpathogenic strains B. cereus ATCC 10987 and B. cereus ATCC 14579. Comparison of these genomes revealed differences in terms of virulence, metabolic competence, structural components, and regulatory mechanisms.

  14. Updates on Clostridium difficile spore biology.

    Science.gov (United States)

    Gil, Fernando; Lagos-Moraga, Sebastián; Calderón-Romero, Paulina; Pizarro-Guajardo, Marjorie; Paredes-Sabja, Daniel

    2017-06-01

    Clostridium difficile is a Gram-positive, anaerobic spore former, and an important nosocomial pathogenic bacterium. C. difficile spores are the morphotype of transmission and recurrence of the disease. The formation of C. difficile spores and their subsequent germination are essential processes during the infection. Recent in vitro and in vivo work has shed light on how spores are formed and the timing of in vivo sporulation in a mouse model. Advances have also been made in our understanding of the machineries involved in spore germination, and how antibiotic-induced dysbiosis affects the metabolism of bile salts and thus impacts C. difficile germination in vivo. Studies have also attempted to identify how C. difficile spores interact with the host's intestinal mucosa. Spore resistance has also been revisited by several groups highlighting the extreme resistance of this morphotype to traditional food processing regimes and disinfectants used in clinical settings. Therefore, the aim of this review is to summarize recent advances on spore formation/germination in vitro and in vivo, spore-host interactions, and spore resistance that contribute to our knowledge of the role of C. difficile spores in the infectious process. Copyright © 2017. Published by Elsevier Ltd.

  15. Survival of B. Horneckiae Spores Under Ground-simulated Space Conditions

    Science.gov (United States)

    Schanche, Bradley

    2012-01-01

    To prevent forward contamination and maintain the scientific integrity of future life detection missions, it is important to characterize and attempt to eliminate terrestrial microorganisms associated with exploratory spacecraft and landing vehicles. Among the organisms isolated from spacecraft-associated habitats, spore-forming microbes are highly resistant to various physical and chemical conditions, which include ionizing and UV radiation, desiccation and oxidative stress, and the harsh environment of outer space or planetary surfaces. Recently a radiation resistant, spore forming bacterial isolate, Bacillus horneckiae, was isolated from a clean room of the Kennedy Space Center where the Phoenix spacecraft was assembled. The exceptionally high tolerance of extreme conditions demonstrated by sporeforming bacteria highlighted the need to assess the viability of these microbes in situ (in real) space. The proposed BOSS (Biofilm Organisms Surfing Space) project aims to understand the mechanisms by which biofilm forming organisms, such as B. horneckiae, will potentially be able to withstand harsh space conditions. As previously stated, the spore producing ability of these species gives them increased survivability to harsh conditions. Some of the spores will have the protective exosporium layer artificially removed before the test to determine if the existence of this layer significantly changes the survivability during the mission. In preparation for that experiment, we analyzed spores which were exposed during a ground simulation, the EXPOSE R2 Biofilm Organisms Surfing Space (BOSS). Previous to exposure, spores were deposited onto spacecraft grade aluminum coupons in a spore suspension calculated to contain between 10(exp 7) and 10(exp 8) spores. This precursor series will be used to establish a baseline survivability function for comparison with the future flight tests during EXPOSE-R. For each coupon, a 10% polyvinyl alcohol (PVA) film was applied and peeled

  16. Compilation of colony forming unit data for Bacillus anthracis and B. atrophaeus before and after exposure to various fogging treatments using peracetic acid or hydrogen peroxide

    Data.gov (United States)

    U.S. Environmental Protection Agency — Data set contains CFU data for positive controls and test coupons for each test, for each material, and for each microorganism used. Also included are efficacy data...

  17. 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.

  18. Safety assessment of the use of Bacillus-based cleaning products.

    Science.gov (United States)

    Berg, Ninna W; Evans, Matthew R; Sedivy, John; Testman, Robert; Acedo, Kimon; Paone, Domenic; Long, David; Osimitz, Thomas G

    2017-11-21

    Non-pathogenic Bacillus species used in cleaning products produce the appropriate enzymes to degrade stains and soils. However, there is little scientific data regarding the human exposure by inhalation of Bacillus spores during or after use of microbial-based cleaning products. Herein, air samples were collected at various locations in a ventilated, carpeted, residential room to determine the air concentration of viable bacteria and spores during and after the application of microbial-based carpet cleaning products containing Bacillus spores. The influence of human activities and vacuuming was investigated. Bioaerosol levels associated with use and post-application activities of whole room carpet treatments were elevated during post-application activity, but quickly returned to the indoor background range. Use of trigger spray spot applications generated aerosolized spores in the immediate vicinity, however, their use pattern and the generation of mostly non-respirable particles suggest minimal risks for pulmonary exposure from their use. The aerosol counts associated with use of these microbial-based cleaners were below the recommendation for safe exposure levels to non-pathogenic and non-toxigenic microorganisms except during application of the spot cleaner. The data presented suggest that carpet cleaning products, containing non-pathogenic Bacillus spores present a low potential for inhalation exposure and consequently minimal risk of adverse effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. 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

  20. PHYLOGENETIC ANALYSIS AND AUTECOLOGY OF SPORE-FORMING BACTERIA FROM HYPERSALINE ENVIRONMENTS.

    Science.gov (United States)

    Gladka, G V; Romanovskaya, V A; Tashyreva, H O; Tashyrev, O B

    2015-01-01

    Multi-resistant to extreme factors spore-forming bacteria of Bacillus genus are isolated from hypersaline environments of the Crimea (Ukraine) and the Dead Sea (Israel). Phylogenetic analysis showed distinction of dominating extremophilic culturable species in studied regions. In Crimean environments they are B. mojavensis and B. simplex, in the Dead Sea ecosystem--B. subtilis subsp. spizizenii, B. subtilis subsp. subtilis, B. licheniformis and B. simplex. Isolates are simultaneously halotolerant and resistant to UV radiation. Strains isolated from the Dead Sea and the Crimea environments were resistant to UV: LD90 and LD99.99 made 100-170 J/m2 and 750-1500 J/m2 respectively. Spores showed higher UV-resistance (LD99.99-2500 J/m2) than the vegetative cells. However the number of spores made 0.02-0.007% of the whole cell population, and should not significantly affect the UV LD99.99 value. Isolates of both environments were halotolerant in the range of 0.1-10% NaCl and thermotolerant in the range of 20-50 °C, and didn't grow at 15 °C. Survival strategy of spore-forming bacteria from hypersaline environments under high UV radiation level can be performed by spore formation which minimize cell damage as well as efficient DNA-repair systems that remove damages.

  1. Ultra-violet-resistant mutants of Bacillus thuringiensis

    International Nuclear Information System (INIS)

    Jones, D.R.; Karunakaran, V.; Hacking, A.J.

    1991-01-01

    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)

  2. Spore liberation in mosses revisited.

    Science.gov (United States)

    Gallenmüller, Friederike; Langer, Max; Poppinga, Simon; Kassemeyer, Hanns-Heinz; Speck, Thomas

    2018-02-01

    The ability to perform hygroscopic movements has evolved in many plant lineages and relates to a multitude of different functions such as seed burial, flower protection or regulation of diaspore release. In most mosses, spore release is controlled by hygroscopic movements of the peristome teeth and also of the spore capsule. Our study presents, for the first time, temporally and spatially well-resolved kinematic analyses of these complex shape changes in response to humidity conditions and provides insights into the sophisticated functional morphology and anatomy of the peristome teeth. In Brachythecium populeum the outer teeth of the peristome perform particularly complex hygroscopic movements during hydration and desiccation. Hydration induces fast inward dipping followed by partial re-straightening of the teeth. In their final shape, wet teeth close the capsule. During desiccation, the teeth perform an outward flicking followed by a re-straightening which opens the capsule. We present a kinematic analysis of these shape changes and of the underlying functional anatomy of the teeth. These teeth are shown to be composed of two layers which show longitudinal gradients in their material composition, structure and geometry. We hypothesize that these gradients result in (i) differences in swelling/shrinking capacity and velocity between the two layers composing the teeth, and in (ii) a gradient of velocity of swelling and shrinking from the tip to the base of the teeth. We propose these processes explain the observed movements regulating capsule opening or closing. This hypothesis is corroborated by experiments with isolated layers of peristome teeth. During hydration and desiccation, changes to the shape and mass of the whole spore capsule accompany the opening and closing. Results are discussed in relation to their significance for humidity-based regulation of spore release.

  3. 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.

  4. Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium.

    Science.gov (United States)

    Todd, Sarah J; Moir, Arthur J G; Johnson, Matt J; Moir, Anne

    2003-06-01

    The exosporium is the outermost layer of spores of Bacillus cereus and its close relatives Bacillus anthracis and Bacillus thuringiensis. For these pathogens, it represents the surface layer that makes initial contact with the host. To date, only the BclA glycoprotein has been described as a component of the exosporium; this paper defines 10 more tightly associated proteins from the exosporium of B. cereus ATCC 10876, identified by N-terminal sequencing of proteins from purified, washed exosporium. Likely coding sequences were identified from the incomplete genome sequence of B. anthracis or B. cereus ATCC 14579, and the precise corresponding sequence from B. cereus ATCC 10876 was defined by PCR and sequencing. Eight genes encode likely structural components (exsB, exsC, exsD, exsE, exsF, exsG, exsJ, and cotE). Several proteins of the exosporium are related to morphogenetic and outer spore coat proteins of B. subtilis, but most do not have homologues in B. subtilis. ExsE is processed from a larger precursor, and the CotE homologue appears to have been C-terminally truncated. ExsJ contains a domain of GXX collagen-like repeats, like the BclA exosporium protein of B. anthracis. Although most of the exosporium genes are scattered on the genome, bclA and exsF are clustered in a region flanking the rhamnose biosynthesis operon; rhamnose is part of the sugar moiety of spore glycoproteins. Two enzymes, alanine racemase and nucleoside hydrolase, are tightly adsorbed to the exosporium layer; they could metabolize small molecule germinants and may reduce the sensitivity of spores to these, limiting premature germination.

  5. Bacillus thuringiensis

    Science.gov (United States)

    Hollensteiner, Jacqueline; Wemheuer, Franziska; Harting, Rebekka; Kolarzyk, Anna M; Diaz Valerio, Stefani M; Poehlein, Anja; Brzuszkiewicz, Elzbieta B; Nesemann, Kai; Braus-Stromeyer, Susanna A; Braus, Gerhard H; Daniel, Rolf; Liesegang, Heiko

    2016-01-01

    Verticillium wilt causes severe yield losses in a broad range of economically important crops worldwide. As many soil fumigants have a severe environmental impact, new biocontrol strategies are needed. Members of the genus Bacillus are known as plant growth-promoting bacteria (PGPB) as well as biocontrol agents of pests and diseases. In this study, we isolated 267 Bacillus strains from root-associated soil of field-grown tomato plants. We evaluated the antifungal potential of 20 phenotypically diverse strains according to their antagonistic activity against the two phytopathogenic fungi Verticillium dahliae and Verticillium longisporum . In addition, the 20 strains were sequenced and phylogenetically characterized by multi-locus sequence typing (MLST) resulting in 7 different Bacillus thuringiensis and 13 Bacillus weihenstephanensis strains. All B. thuringiensis isolates inhibited in vitro the tomato pathogen V. dahliae JR2, but had only low efficacy against the tomato-foreign pathogen V. longisporum 43. All B. weihenstephanensis isolates exhibited no fungicidal activity whereas three B. weihenstephanensis isolates showed antagonistic effects on both phytopathogens. These strains had a rhizoid colony morphology, which has not been described for B. weihenstephanensis strains previously. Genome analysis of all isolates revealed putative genes encoding fungicidal substances and resulted in identification of 304 secondary metabolite gene clusters including 101 non-ribosomal polypeptide synthetases and 203 ribosomal-synthesized and post-translationally modified peptides. All genomes encoded genes for the synthesis of the antifungal siderophore bacillibactin. In the genome of one B. thuringiensis strain, a gene cluster for zwittermicin A was detected. Isolates which either exhibited an inhibitory or an interfering effect on the growth of the phytopathogens carried one or two genes encoding putative mycolitic chitinases, which might contribute to antifungal activities

  6. Effects of steam autoclave treatment on Geobacillus stearothermophilus spores.

    Science.gov (United States)

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

    2016-11-01

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

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

    Science.gov (United States)

    Gopal, Selvakumar; Shagol, Charlotte C; Kang, Yeongyeong; Chung, Bong Nam; Han, Seung Gab; Tong-Min, Sa

    2018-02-02

    The propagation of pure cultures of AMF is an essential requirement for their large scale agricultural application and commercialization as biofertilizers. The present study aimed to propagate AMF using the single spore inoculation technique and compare their propagation ability with the known reference spores. Arbuscular mycorrhizal fungal spores were collected from the salt-affected Saemangeum reclaimed soil in South Korea. The technique involved inoculation of Sorghum-Sudan grass (Sorghum bicolor L.) seedlings with single, healthy spores on filter paper followed by the transfer of successfully colonized seedlings to 1 kg capacity pots containing sterilized soil. After the first plant cycle, the contents were transferred to 2.5 kg capacity pots containing sterilized soil. Among the 150 inoculants, only 27 seedlings were colonized by AMF spores. After 240 days, five inoculants among the 27 seedlings resulted in the production of over 500 spores. The 18S rDNA sequencing of spores revealed that the spores produced through single spore inoculation method belonged to Gigaspora margarita, Claroideoglomus lamellosum, and Funneliformis mosseae. Furthermore, indigenous spore Funneliformis mosseae M-1 reported a higher spore count than the reference spores. The AMF spores produced using single spore inoculation technique may serve as potential bio-inoculants with an advantage of being more readily adopted by farmers due to the lack of requirement of a skilled technique in spore propagation. The results of the current study describes the feasible and cost effective method to mass produce AMF spores for large scale application. The AMF spores obtained from this method can effectively colonize plant roots and may be easily introduced to the new environment. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  8. Evaluation of the Sporicidal Activity of Ethanol Extract of Arctium lappa Root against Bacillus cereus

    Directory of Open Access Journals (Sweden)

    Vajihe Karbasizade

    2014-10-01

    Full Text Available Background: Bacillus cereus is one of the most common causes of food spoilage, keratitis, endophthalmitis, and panophthalmitis. These bacteria produce spores which are resistant to chemical and physical agents. Nowadays, the sporicidal properties of plants have been considered as alternatives to chemical sporicidal agents. Materials and Methods: In this empirical-experimental study the effect of ethanol extract of edible burdock (Arctium lappa root has been studied on Bacillus cereus spores. In this investigation, the suspensions of tested microorganisms were cultured in sporulating agar. Sporulation process was assessed by optical microscopy following the staining of spores. Then the produced spores were exposed to various concentrations (100, 150, 200, 250, 300 mg/mL of ethanol extract of edible burdock (Arctium lappa root and finally the remaining spores were counted. With increasing concentrations of ethanol extract, the number of spores declined. Results: Pearson correlation showed inverse relation between the spores count and concentration of ethanol extract of edible burdock (Arctium lappa root (r=-0.765, p<0.001. The most effective extract concentration was 300 mg /mL. Conclusion: Ethanol extract of edible burdock (Arctium lappa root, has sporicidal activity. Only, the sporicidal nature of ethanol extract has been evaluated by this study; therefore, the assessment of other extracts and essences is necessary.

  9. Bacillus As Potential Probiotics: Status, Concerns, and Future Perspectives

    Directory of Open Access Journals (Sweden)

    Fouad M. F. Elshaghabee

    2017-08-01

    Full Text Available Spore-forming bacilli are being explored for the production and preservation of food for many centuries. The inherent ability of production of large number of secretory proteins, enzymes, antimicrobial compounds, vitamins, and carotenoids specifies the importance of bacilli in food chain. Additionally, Bacillus spp. are gaining interest in human health related functional food research coupled with their enhanced tolerance and survivability under hostile environment of gastrointestinal tract. Besides, bacilli are more stable during processing and storage of food and pharmaceutical preparations, making them more suitable candidate for health promoting formulations. Further, Bacillus strains also possess biotherapeutic potential which is connected with their ability to interact with the internal milieu of the host by producing variety of antimicrobial peptides and small extracellular effector molecules. Nonetheless, with proposed scientific evidences, commercial probiotic supplements, and functional foods comprising of Bacillus spp. had not gained much credential in general population, since the debate over probiotic vs pathogen tag of Bacillus in the research and production terrains is confusing consumers. Hence, it’s important to clearly understand the phenotypic and genotypic characteristics of selective beneficial Bacillus spp. and their substantiation with those having GRAS status, to reach a consensus over the same. This review highlights the probiotic candidature of spore forming Bacillus spp. and presents an overview of the proposed health benefits, including application in food and pharmaceutical industry. Moreover, the growing need to evaluate the safety of individual Bacillus strains as well as species on a case by case basis and necessity of more profound analysis for the selection and identification of Bacillus probiotic candidates are also taken into consideration.

  10. Raman spectroscopy of Bacillus thuringiensis physiology and inactivation

    Science.gov (United States)

    Morrow, J. B.; Almeida, J.; Cole, K. D.; Reipa, V.

    2012-12-01

    The ability to detect spore contamination and inactivation is relevant to developing and determining decontamination strategy success for food and water safety. This study was conducted to develop a systematic comparison of nondestructive vibrational spectroscopy techniques (Surface-Enhanced Raman Spectroscopy, SERS, and normal Raman) to determine indicators of Bacillus thuringiensis physiology (spore, vegetative, outgrown, germinated and inactivated spore forms). SERS was found to provide better resolution of commonly utilized signatures of spore physiology (dipicolinic acid at 1006 cm-1 and 1387 cm-1) compared to normal Raman and native fluorescence indigenous to vegetative and outgrown cell samples was quenched in SERS experiment. New features including carotenoid pigments (Raman features at 1142 cm-1, 1512 cm-1) were identified for spore cell forms. Pronounced changes in the low frequency region (300 cm-1 to 500 cm-1) in spore spectra occurred upon germination and inactivation (with both free chlorine and by autoclaving) which is relevant to guiding decontamination and detection strategies using Raman techniques.

  11. Construction of Bacillus thuringiensis Simulant Strains Suitable for Environmental Release.

    Science.gov (United States)

    Park, Sangjin; Kim, Changhwan; Lee, Daesang; Song, Dong Hyun; Cheon, Ki Cheol; Lee, Hong Suk; Kim, Seong Joo; Kim, Jee Cheon; Lee, Sang Yup

    2017-05-01

    For a surrogate bacterium to be used in outdoor studies, it is important to consider environmental and human safety and ease of detection. Recently, Bacillus thuringiensis , a popular bioinsecticide bacterium, has been gaining attention as a surrogate bacterium for use in biodefense. In this study, we constructed simulant strains of B. thuringiensis with enhanced characteristics for environmental studies. Through transposon mutagenesis, pigment genes were inserted into the chromosome, producing yellow-colored colonies for easy detection. To prevent persistence of spores in the environment, a genetic circuit was designed to produce a spore without sporulation capability. Two loxP sites were inserted, one on each side of the spo0A gene, which encodes a sporulation master regulator, and a sporulation-dependent Cre expression cassette was inserted into the chromosome. This genetic circuit successfully deleted spo0A during sporulation, producing spores that lacked the spo0A gene. In addition, two major α/β-type small acid-soluble spore protein (SASP) genes, predicted by synteny analysis, were deleted. The spores of the mutant strain showed increased UV-C sensitivity and quickly lost viability when tested in a solar simulator. When the spores of the mutant strain were administered to the lungs of BALB/c mice, cells were quickly removed from the body, suggesting enhanced in vivo safety. All strains constructed in this study contain no antibiotic resistance markers and all heterologous genes were inserted into the chromosome, which are useful features for simulants to be released into the environment. IMPORTANCE B. thuringiensis has recently been receiving increasing attention as a good spore simulant in biodefense research. However, few studies were done to properly address many important features of B. thuringiensis as a simulant in environmental studies. Since spores can persist in the environment for years after release, environmental contamination is a big problem

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

    Science.gov (United States)

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

    2012-11-01

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

  13. The solar UV environment and bacterial spore UV resistance: considerations for Earth-to-Mars transport by natural processes and human spaceflight

    Science.gov (United States)

    Nicholson, Wayne L.; Schuerger, Andrew C.; Setlow, Peter

    2005-01-01

    The environment in space and on planets such as Mars can be lethal to microorganisms because of the high vacuum and high solar radiation flux, in particular UV radiation, in such environments. Spores of various Bacillus species are among the organisms most resistant to the lethal effects of high vacuum and UV radiation, and as a consequence are of major concern for planetary contamination via unmanned spacecraft or even natural processes. This review focuses on the spores of various Bacillus species: (i) their mechanisms of UV resistance; (ii) their survival in unmanned spacecraft, space flight and simulated space flight and Martian conditions; (iii) the UV flux in space and on Mars; (iv) factors affecting spore survival in such high UV flux environments.

  14. The solar UV environment and bacterial spore UV resistance: considerations for Earth-to-Mars transport by natural processes and human spaceflight

    International Nuclear Information System (INIS)

    Nicholson, Wayne L.; Schuerger, Andrew C.; Setlow, Peter

    2005-01-01

    The environment in space and on planets such as Mars can be lethal to microorganisms because of the high vacuum and high solar radiation flux, in particular UV radiation, in such environments. Spores of various Bacillus species are among the organisms most resistant to the lethal effects of high vacuum and UV radiation, and as a consequence are of major concern for planetary contamination via unmanned spacecraft or even natural processes. This review focuses on the spores of various Bacillus species: (i) their mechanisms of UV resistance; (ii) their survival in unmanned spacecraft, space flight and simulated space flight and Martian conditions; (iii) the UV flux in space and on Mars; (iv) factors affecting spore survival in such high UV flux environments

  15. Morpho-structural variations of bacterial spores after treatment in steam vacuum assisted autoclave.

    Science.gov (United States)

    Fonzi, M; Montomoli, E; Gasparini, R; Devanna, D; Fonzi, L

    1999-01-01

    This study intended to verify, through microbiological techniques and TEM investigations, the killing of bacterial spores after treatment in steam autoclave, and to propose strictly morphological considerations about the target of this sterilisation process. Autoclave is the most common device for sterilising instruments in order to prevent cross infections in dental offices. The autoclave efficiency has been improved in the last years and part of this improvement is related to both a better and more correct use of the autoclave system and to the technological innovations introduced in the last generation of devices. However, associations as ADA or CDC suggest to regularly verify the process of 'autoclaving' through biological indicators (BI). The most commonly used BI are made of spores strips or suspensions of Bacillus Subtilis (pb 168) and Bacillus Stearothermophilus (ATCC 10149). They visually prove, changing colours on enzymatic base, the death of micro-organism and if the physical parameters, necessary for sterilisation, have been achieved. These two strains of endospore-forming bacteria were processed and prepared following two different techniques: Karnovsky fixed and epon embedded--phosphotungstic acid fixed for direct observation. The kind and the extent of analysed modifications are extremely various: from deep lacerations, which changed the spore structure, to little clefts which let the cytoplasm go out.

  16. Metabolism and the triggering of germination of Bacillus megaterium

    International Nuclear Information System (INIS)

    Scott, I.R.; Ellar, D.J.

    1978-01-01

    L-[2,3- 3 H]Alanine was used to probe for metabolism of alanine during triggering of germination of spores of Bacillus megaterium KM. No detectable incorporation of label into any compound, including water, was found, indicating that any metabolism involving the alanine germinant must be at a very low rate and also that alanine racemase is absent from spores of this strain. Spores were germinated in 3 H 2 0 to find if any of the many metabolic reactions causing irreversible incorporation of 3 H into reaction products took place during triggering og germination. No incorporation was detected until 2-3 min after addition of germinants. It is therefore concluded that a wide variety of metabolic routes, including glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and amino acid metabolism are either not involved in the reactions causing the triggering of germination or operate at an extremely low rate during this process. (author)

  17. Inhibition of Bacillus cereus in milk fermented with kefir grains.

    Science.gov (United States)

    Kakisu, Emiliano J; Abraham, Analía G; Pérez, Pablo F; De Antoni, Graciela L

    2007-11-01

    The effects of kefir-fermented milk were tested against a toxigenic strain of Bacillus cereus. The incubation of milk with B. cereus spores plus 5% kefir grains prevented spore germination and growth of vegetative forms. In contrast, when 1% kefir grains was used, no effects were observed. The presence of metabolically active kefir grains diminished titers of nonhemolytic enterotoxin A, as assessed by enzyme-linked immunosorbent assay. During fermentation, kefir microorganisms produce extracellular metabolites such as organic acids, which could play a role in the inhibition of spore germination and growth of B. cereus, although the effect of other factors cannot be ruled out. Results of the present study show that kefir-fermented milk is able to antagonize key mechanisms involved in the growth of B. cereus as well as interfere with the biological activity of this microorganism.

  18. Antifungal activity of indigenous Bacillus spp. isolated from soil

    Directory of Open Access Journals (Sweden)

    Bjelić Dragana Đ.

    2017-01-01

    Full Text Available Biocontrol using plant growth-promoting rhizobacteria (PGPR represents an alternative approach to disease management, since PGPR are known to promote growth and reduce diseases in various crops. Among the different PGPR, members of the genus Bacillus are prefered for most biotechnological uses due to their capability to form extremely resistant spores and produce a wide variety of metabolites with antimicrobial activity. The objective of this research was to identify antagonistic bacteria for management of the plant diseases. Eleven isolates of Bacillus spp. were obtained from the soil samples collected from different localities in the Province of Vojvodina. The antifungal activity of bacterial isolates against five fungal species was examined using a dual plate assay. Bacillus isolates exhibited the highest antifungal activity against Fusarium proliferatum, Fusarium oxysporum f. sp. cepae and Alternaria padwickii, while they had the least antagonistic effect on Fusarium verticillioides and Fusarium graminearum. Molecular identification showed that effective bacterial isolates were identified as Bacillus safensis (B2, Bacillus pumilus (B3, B11, Bacillus subtilis (B5, B7 and Bacillus megaterium (B8, B9. The highest antagonistic activity was exhibited by isolates B5 (from 39% to 62% reduction in fungal growth and B7 (from 40% to 71% reduction in fungal growth. These isolates of B. subtilis could be used as potential biocontrol agents of plant diseases. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. TR-31073

  19. Heavy metals and their radionuclides uptake by Bacillus Licheniformis

    International Nuclear Information System (INIS)

    Ramadan, A.A.; Ahmed, M.M.; Abo-state, M.A.M.; Sarhan, M.; Faroqe, M.

    2007-01-01

    Bacillus licheniformis is a gram positive spore forming bacterium. Different concentrations of cobalt affected the ability of Co uptake and growth of Bacillus licheniformis. As the concentration increased, both the uptake and growth were decreased. Maximum Co uptake was found at ph 7.0, while for growth was ph 8.0. The optimum temperature for uptake and growth was 40 degree C and 20% inoculum size represents the maximum cobalt uptake by Bacillus licheniformis. Also, maximum uptake was recorded after 72 hours, incubation period. As the concentration of cesium was increased till 400 mg/l, the uptake was also increased. The optimum cesium uptake and growth was at ph 8.0. The optimum growth was at 45 degree C while Cs uptake was found at 35 degree C and 15% inoculum size represented the maximum Cs uptake. After 72 hour incubation period, maximum Cs uptake was recorded. Generally, Bacillus licheniformis removed more than 80% of Co and 50% of Cs from the broth medium. Addition of clay to Bacillus licheniformis increased both Co or Cs uptake. Bacillus licheniformis was gamma resistant and 10 KGy reduced the viability by 5.3 log cycles. The irradiated and non-irradiated cultures can grow on 500 or 700 mg Co or Cs. Bacillus licheniformis removed 99.32% of the Co radionuclides and 99.28% of Cs radionuclides

  20. Real-Time PCR Identification of Unique Bacillus anthracis Sequences.

    Science.gov (United States)

    Cieślik, P; Knap, J; Kolodziej, M; Mirski, T; Joniec, J; Graniak, G; Zakowska, D; Winnicka, I; Bielawska-Drózd, A

    2015-01-01

    Bacillus anthracis is a spore-forming, Gram-positive microorganism. It is a causative agent of anthrax, a highly infectious disease. It belongs to the "Bacillus cereus group", which contains other closely related species, including Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus pseudomycoides. B. anthracis naturally occurs in soil environments. The BA5345 genetic marker was used for highly specific detection of B. anthracis with TaqMan probes. The detection limit of a real-time PCR assay was estimated at the level of 16.9 copies (CI95% - 37.4 to 37.86, SD = 0.2; SE = 0.118). Oligonucleotides designed for the targeted sequences (within the tested locus) revealed 100 % homology to B. anthracis strain reference sequences deposited in the database (NCBI) and high specificity to all tested B. anthracis strains. Additional in silico analysis of plasmid markers pag and cap genes with B. anthracis strains included in the database was carried out. Our study clearly indicates that the BA5345 marker can be used with success as a chromosomal marker in routine identification of B. anthracis; moreover, detection of plasmid markers indicates virulence of the examined strains.

  1. Validated modified Lycopodium spore method development for ...

    African Journals Online (AJOL)

    Validated modified lycopodium spore method has been developed for simple and rapid quantification of herbal powdered drugs. Lycopodium spore method was performed on ingredients of Shatavaryadi churna, an ayurvedic formulation used as immunomodulator, galactagogue, aphrodisiac and rejuvenator. Estimation of ...

  2. What can spores do for us?

    NARCIS (Netherlands)

    Wolken, W.A.M.; Tramper, J.; Werf, van der M.J.

    2003-01-01

    Many organisms have the ability to form spores, a remarkable phase in their life cycles. Compared with vegetative cells, spores have several advantages (e.g. resistance to toxic compounds, temperature, desiccation and radiation) making them well suited to various applications. The applications of

  3. Sphagnum moss disperses spores with vortex rings.

    Science.gov (United States)

    Whitaker, Dwight L; Edwards, Joan

    2010-07-23

    Sphagnum spores, which have low terminal velocities, are carried by turbulent wind currents to establish colonies many kilometers away. However, spores that are easily kept aloft are also rapidly decelerated in still air; thus, dispersal range depends strongly on release height. Vascular plants grow tall to lift spores into sufficient wind currents for dispersal, but nonvascular plants such as Sphagnum cannot grow sufficiently high. High-speed videos show that exploding capsules of Sphagnum generate vortex rings to efficiently carry spores high enough to be dispersed by turbulent air currents. Spores launched ballistically at similar speeds through still air would travel a few millimeters and not easily reach turbulent air. Vortex rings are used by animals; here, we report vortex rings generated by plants.

  4. Ptaquiloside in bracken spores from Britain.

    Science.gov (United States)

    Rasmussen, Lars Holm; Schmidt, Bjørn; Sheffield, Elizabeth

    2013-03-01

    Secondary metabolites from bracken fern (Pteridium aquilinum (L.) Kuhn) are suspected of causing cancer in humans. The main carcinogen is the highly water-soluble norsesquiterpene glucoside ptaquiloside, which may be ingested by humans through food, e.g. via contaminated water, meat or milk. It has been postulated that carcinogens could also be ingested through breathing air containing bracken spores. Ptaquiloside has not previously been identified in bracken spores. The aim of the study was to determine whether ptaquiloside is present in bracken spores, and if so, to estimate its content in a collection of spores from Britain. Ptaquiloside was present in all samples, with a maximum of 29 μg g(-1), which is very low compared to other parts of the fern. Considering the low abundance of spores in breathing air under normal conditions, this exposure route is likely to be secondary to milk or drinking water. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Complete Genome Sequence of Bacillus horikoshii Strain 20a from Cuatro Cienegas, Coahuila, Mexico

    OpenAIRE

    Zarza, Eugenia; Alcaraz, Luis D.; Aguilar-Salinas, Bernardo; Islas, Africa; Olmedo-?lvarez, Gabriela

    2017-01-01

    ABSTRACT We sequenced the Bacillus horikoshii 20a genome, isolated from sediment collected in Cuatro Cienegas, Mexico. We identified genes involved in establishing antagonistic interactions in microbial communities (antibiotic resistance and bacteriocins) and genes related to the metabolism of cyanophycin, a reserve compound and spore matrix material potentially relevant for survival in an oligotrophic environment.

  6. A new chemically defined medium for the growth and sporulation of Bacillus cereus strains in anaerobiosis.

    Science.gov (United States)

    Abbas, Amina Aicha; Planchon, Stella; Jobin, Michel; Schmitt, Philippe

    2014-10-01

    A new chemically defined liquid medium, MODS, was developed for the aerobic growth and anaerobic growth and sporulation of Bacillus cereus strains. The comparison of sporulation capacity of 18 strains of B. cereus has shown effective growth and spore production in anaerobiosis.. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Nuclear and cell division in Bacillus subtilis. Antibiotic-induced morphological changes

    NARCIS (Netherlands)

    van Iterson, W.; Aten, J. A.

    1976-01-01

    Incubation of Bacillus subtilis after outgrowth from spores in the presence of four different antibiotics in two different concentrations, showed that septation can occur without termination of nuclear division. Septation is then only partially uncoupled from the normal division cycle. Observations

  8. Phosphatases modulate the bistable sporulation gene expression pattern in Bacillus subtilis

    NARCIS (Netherlands)

    Veening, JW; Hamoen, LW; Kuipers, OP

    Spore formation in the Gram- positive bacterium Bacillus subtilis is a last resort adaptive response to starvation. To initiate sporulation, the key regulator in this process, Spo0A, needs to be activated by the so-called phosphorelay. Within a sporulating culture of B. subtilis, some cells initiate

  9. Immune modulation by Bacillus subtilus-based direct-fed microbials in commercial broiler chickens

    Science.gov (United States)

    Direct-fed microbials (DFMs), also known as probiotics, have been successfully used to improve the balance of gut microbiota. Spores of Bacillus subtilis, have been used as DFMs for food animals and humans and our previous studies showed that dietary supplementation of broiler chickens with a B. su...

  10. Germinant receptor diversity and germination responses of four strains of the Bacillus cereus group

    NARCIS (Netherlands)

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

    2010-01-01

    Four strains of the Bacillus cereus group were compared for their germinant receptor composition and spore germination capacity. Phylogenetic analysis of the germinant receptor encoding operons of the enterotoxic strains B. cereus ATCC 14579 and ATCC 10987, the emetic strain AH187, and the

  11. Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production

    Science.gov (United States)

    Spore-forming Bacillus strains that produce extracellular poly-'-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 365 strains, including B. subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting n...

  12. Beetroot-Pigment-Derived Colorimetric Sensor for Detection of Calcium Dipicolinate in Bacterial Spores

    Science.gov (United States)

    Gonçalves, Letícia Christina Pires; Da Silva, Sandra Maria; DeRose, Paul C.; Ando, Rômulo Augusto; Bastos, Erick Leite

    2013-01-01

    In this proof-of-concept study, we describe the use of the main red beet pigment betanin for the quantification of calcium dipicolinate in bacterial spores, including Bacillus anthracis. In the presence of europium(III) ions, betanin is converted to a water-soluble, non-luminescent orange 1∶1 complex with a stability constant of 1.4×105 L mol–1. The addition of calcium dipicolinate, largely found in bacterial spores, changes the color of the aqueous solution of [Eu(Bn)+] from orange to magenta. The limit of detection (LOD) of calcium dipicolinate is around 2.0×10–6 mol L–1 and the LOD determined for both spores, B. cereus and B. anthracis, is (1.1±0.3)×106 spores mL–1. This simple, green, fast and low cost colorimetric assay was selective for calcium dipicolinate when compared to several analogous compounds. The importance of this work relies on the potential use of betalains, raw natural pigments, as colorimetric sensors for biological applications. PMID:24019934

  13. Effect of oral administration of Bacillus coagulans B37 and Bacillus pumilus B9 strains on fecal coliforms, Lactobacillus and Bacillus spp. in rat animal model

    Directory of Open Access Journals (Sweden)

    Lopamudra Haldar

    2016-07-01

    Full Text Available Aim: To investigate the effect of oral administration of two Bacillus strains on fecal coliforms, Lactobacillus and Bacillus spp. in rat animal model. Materials and Methods: An in vivo experiment was conducted for 49-day period on 36 adult male albino Wister rats divided equally into to four groups. After 7-day adaptation period, one group (T1 was fed on sterile skim milk along with basal diet for the next 28 days. Second (T2 and (T3 groups received spore biomass of Bacillus coagulans B37 and Bacillus pumilus B9, respectively, suspended in sterilized skim milk at 8-9 log colony-forming units/ml plus basal diet for 28 days, while control group (T4 was supplied with clean water along with basal diet. There was a 14-day post-treatment period. A total of 288 fecal samples (8 fecal collections per rat were collected at every 7-day interval starting from 0 to 49 days and subjected to the enumeration of the counts of coliforms and lactobacilli and Bacillus spores using respective agar media. In vitro acid and bile tolerance tests on both the strains were performed. Results: The rats those (T2 and T3 received either B. coagulans B37 or B. pumilus B9 spore along with non-fermented skim milk showed decrease (p<0.01 in fecal coliform counts and increase (p<0.05 in both fecal lactobacilli and Bacillus spore counts as compared to the control group (T4 and the group fed only skim milk (T1. In vitro study indicated that both the strains were found to survive at pH 2.0 and 3.0 even up to 3 h and tolerate bile up to 2.0% concentration even after 12 h of exposure. Conclusions: This study revealed that oral administration of either B. coagulans B37 or B. pumilus B9 strains might be useful in reducing coliform counts accompanied by concurrent increase in lactobacilli counts in the intestinal flora in rats.

  14. Bacillus amyloliquefaciens

    Science.gov (United States)

    Qin, Yuxuan; Shang, Qingmao; Zhang, Ying; Li, Pinglan; Chai, Yunrong

    2017-01-01

    Vegetable plug seedling has become the most important way to produce vegetable seedlings in China. This seedling method can significantly improve the quality and yield of vegetables compared to conventional methods. In the process of plug seedling, chemical fertilizers or pesticides are often used to improve the yield of the seedlings albeit with increasing concerns. Meanwhile, little is known about the impact of beneficial bacteria on the rhizosphere microbiota and the growth conditions of vegetables during plug seedling. In this study, we applied a culture-independent next-generation sequencing-based approach and investigated the impact of a plant beneficial bacterium, Bacillus amyloliquefaciens L-S60, on the composition and dynamics of rhizosphere microbiota and the growth conditions of cucumbers during plug seedling. Our results showed that application of L-S60 significantly altered the structure of the bacterial community associated with the cucumber seedling; presence of beneficial rhizosphere species such as Bacillus, Rhodanobacter, Paenibacillus, Pseudomonas, Nonomuraea , and Agrobacterium was higher upon L-S60 treatment than in the control group. We also measured the impact of L-S60 application on the physiological properties of the cucumber seedlings as well as the availability of main mineral elements in the seedling at different time points during the plug seedling. Results from those measurements indicated that L-S60 application promoted growth conditions of cucumber seedlings and that more available mineral elements were detected in the cucumber seedlings from the L-S60 treated group than from the control group. The findings in this study provided evidence for the beneficial effects of plant growth-promoting rhizosphere bacteria on the bacterial community composition and growth conditions of the vegetables during plug seedling.

  15. Comparative study on disinfection potency of spore forming bacteria by electron-beam irradiation and gamma-ray irradiation

    International Nuclear Information System (INIS)

    Takizawa, Hironobu; Suzuki, Satoru; Suzuki, Tetsuya; Takama, Kozo; Hayashi, Toru; Yasumoto, Kyoden.

    1990-01-01

    Along with gamma-ray irradiation, electron-beam irradiation (EB) is a method to disinfect microorganisms which cause food decomposition and food-poisoning. The present study was undertaken to compare sterilization efficacy of EB and gamma-ray irradiation on bacterial spores and vegetative cells under various conditions. Spores of Bacillus pumilus, a marker strain for irradiation study, and Bacillus stearothermophilus known as a thermophilic bacteria were irradiated by electron-beam and gamma-ray separately at irradiation dose of 0 to 10 kGy on combination of wet/dry and aerobic/anaerobic conditions. Sterilization effect of irradiation on spores was evaluated by colony counting on agar plates. Results showed that both EB and gamma-ray irradiation gave sufficient sterilization effect on spores, and the sterilization effect increased exponentially with irradiation dose. The sterilization effect of gamma-ray irradiation was higher than that of EB in all cases. Higher disinfection effect was observed under aerobic condition. The present study suggests that oxygen supply in EB is more important than gamma-ray irradiation. No results suggesting that chlorine ion at 0.1 ppm (as available chlorine concentration) enhanced the sterilization efficacy of either EB or gamma-ray irradiation was obtained under any conditions examined. (author)

  16. «KING OF PROBIOTICS» BACILLUS COAGULANS IN MODERN COMBINED PROBIOTIC PREPARATIONS LAKTOVIT FORTE (FULL REVIEW

    Directory of Open Access Journals (Sweden)

    Bomko TV

    2016-03-01

    Full Text Available Bacillus coagulans has an advantage over most other bacteria used as probiotics. It occupies an intermediate position between the genera Bacillusand Lactobacillus, is a spore-forming bacteria that produce lactic acid.This bacteria in the spores form can tolerate well technology processes, resistant to antibiotics and antiseptics, does not collapse under the influence of gastric juice and bile. Getting into the duodenum, the spores germinate into vegetative forms and begin vegetation and growth, providing probiotic effects.Bacillus coagulans refers to semi-residental bacteria - performing in the human probiotic function, it passes the sporulation phase and slowly leaves the body, standing out in the faeces in the spores form. Thus, it does not violate the personal composition of intestinal microflora.Probiotic Bacillus coagulans enhances the microbiological composition of the intestine, increasing the number of obligate microorganisms and displacing pathogenic flora. Mechanisms of this action based on the lactic acid production and some bacteriocins synthesis, also on the immunomodulatory effect - stimulation of cellular and humoral immunity. The bacterial cell wall and spores are the main immunomodulatory compounds of the Bacillus coagulans.Apparently, namely Bacillus coagulans immunomodulatory properties play a crucial role in the pharmacological effects. It is now well known about the important role of immune system in the pathogenesis of many diseases; it has the clinical effect without the need for intensive growth of bacteria and intestinal colonization; even small amounts of spores are sufficient for pharmacological effect; many experimental evidences of the spore penetration into the lymphatic system and interaction with immunocompetent cells, as well as local and systemic immune effects of probiotic.In addition to this main action, Bacillus coagulans helps to digest lactose, possesses anti-inflammatory and antioxidant activity, as well

  17. Measurements of the Ultraviolet Fluorescence Cross Sections and Spectra of Bacillus Anthracis Simulants

    Energy Technology Data Exchange (ETDEWEB)

    Stephens, J.R.

    1998-09-01

    Measurements of the ultraviolet autofluorescence spectra and absolute cross sections of the Bacillus anthracis (Ba) simulants Bacillus globigii (Bg), Bacillus megaterium (Bm), Bacillus subtilis (Bs), and Bacillus cereus (Bc) were measured. Fluorescence spectra and cross sections of pine pollen (Pina echinata) were measured for comparison. Both dried vegetative cells and spores separated from the sporulated vegetative material were studied. The spectra were obtained by suspending a small number (<10) of particles in air in our Single Particle Spectroscopy Apparatus (SPSA), illuminating the particles with light from a spectrally filtered arc lamp, and measuring the fluorescence spectra of the particles. The illumination was 280 nm (20 nm FWHM) and the fluorescence spectra was measured between 300 and 450 nm. The fluorescence cross section of vegetative Bg peaks at 320 nm with a maximum cross section of 5 X 10{sup -14} cm{sup 2}/sr-nm-particle while the Bg spore fluorescence peaks at 310 nm with peak fluorescence of 8 X 10{sup -15} cm{sup 2}/sr-nm-particle. Pine pollen particles showed a higher fluorescence peaking at 355 nm with a cross section of 1.7 X 10{sup -13} cm{sup 2}/sr-nm-particle. Integrated cross sections ranged from 3.0 X 10{sup -13} for the Bg spores through 2.25 X 10{sup -12} (cm{sup 2}/sr-particle) for the vegetative cells.

  18. Hygiene Aspects of the Biogas Process with Emphasis on Spore-Forming Bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Bagge, Elisabeth

    2009-07-01

    Biogas is a renewable source of energy which can be obtained from processing of biowaste. The digested residues can be used as fertiliser. Biowaste intended for biogas production contains pathogenic micro-organisms. A pre-pasteurisation step at 70 deg C for 60 min before anaerobic digestion reduces non spore-forming bacteria such as Salmonella spp. To maintain the standard of the digested residues it must be handled in a strictly hygienic manner to avoid recontamination and re-growth of bacteria. The risk of contamination is particularly high when digested residues are transported in the same vehicles as the raw material. However, heat treatment at 70 deg C for 60 min will not reduce spore-forming bacteria such as Bacillus spp. and Clostridium spp. Spore-forming bacteria, including those that cause serious diseases, can be present in substrate intended for biogas production. The number of species and the quantity of Bacillus spp. and Clostridium spp. in manure, slaughterhouse waste and in samples from different stages during the biogas process were investigated. The number of species of clostridia seemed to decrease following digestion, likewise the quantity. However, Bacillus spp. seemed to pass unaffected through the biogas process. In laboratory-scale experiments the effects on clostridia during pasteurisation and digestion were investigated. Pathogenic clostridia were inoculated in substrates from homogenisation tanks and digester tanks. The inoculated clostridia remained after pasteurisation, but the impacts of digestion differ between different species. Culture followed by identification of C. chauvoei by PCR in samples from cattle died from blackleg, is faster and safer than culture followed by biochemical identification of C. chauvoei. However, for environmental samples the PCR method is not practically applicable for detection of C. chauvoei. To avoid spreading of diseases via biogas plants when digested residues are spread on arable land, a pasteurisation

  19. Bacillus cereus food poisoning: international and Indian perspective.

    Science.gov (United States)

    Tewari, Anita; Abdullah, Swaid

    2015-05-01

    Food borne illnesses result from eating food or drinking beverages that are contaminated with chemical matter, heavy metals, parasites, fungi, viruses and Bacteria. Bacillus cereus is one of the food-borne disease causing Bacteria. Species of Bacillus and related genera have long been troublesome to food producers on account of their resistant endospores. Their spores may be present on various types of raw and cooked foods, and their ability to survive high cooking temperatures requires that cooked foods be served hot or cooled rapidly to prevent the growth of this bacteria. Bacillus cereus is well known as a cause of food poisoning, and much more is now known about the toxins produced by various strains of this species, so that its significance in such episodes are clearer. However, it is still unclear why such cases are so rarely reported worldwide.

  20. Persistence of Bacillus thuringiensis bioinsecticides in the gut of human-flora-associated rats

    DEFF Research Database (Denmark)

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

    2006-01-01

    The capability of two bioinsecticide strains of Bacillus thuringiensis (ssp. israelensis and ssp. kurstaki) to germinate and persist in vivo in the gastrointestinal tract of human-flora-associated rats was studied. Rats were dosed either with vegetative cells or spores of the bacteria for 4...... consecutive days. In animals fed spores, B. thuringiensis cells were detected in faecal and intestinal samples of all animals, whereas vegetative cells only poorly survived the gastric passage. Heat-treatment of intestinal samples, which kills vegetative cells, revealed that B. thuringiensis spores were...... capable of germination in the gastrointestinal tract. In one animal fed spores of B. thuringiensis ssp. kurstaki, these bacteria were detected at high density (10(3)-10(4) CFU g(-1) faecal and intestinal samples) even 2 weeks after the last dosage. In the same animal, passage of B. thuringiensis ssp...

  1. Identification and characterization of clinical Bacillus spp. isolates phenotypically similar to Bacillus anthracis.

    Science.gov (United States)

    Beesley, Cari A; Vanner, Cynthia L; Helsel, Leta O; Gee, Jay E; Hoffmaster, Alex R

    2010-12-01

    Bacillus anthracis, the etiological agent of anthrax, is a gram-positive, spore-forming rod, with colonies exhibiting a unique ground-glass appearance, and lacking hemolysis and motility. In addition to these phenotypes, several others traits are characteristic of B. anthracis such as susceptibility to gamma phage, the presence of two virulence plasmids (pX01 and pX02), and specific cell wall and capsular antigens that are commonly detected by direct fluorescent-antibody assays. We report on the identification and characterization of 14 Bacillus megaterium and four Bacillus sp. clinical isolates that are nonhemolytic, nonmotile, and produce a capsule antigenically similar to B. anthracis. This work furthers our understanding of Bacillus diversity and the limitations of the assays and phenotypes that are used to differentiate species in this genus. Further work is necessary to understand whether these strains are opportunistic pathogens or just contaminates. FEMS Microbiology Letters © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. No claim to original US government works.

  2. Germinant receptor diversity and germination responses of four strains of the Bacillus cereus group.

    Science.gov (United States)

    van der Voort, Menno; García, Diego; Moezelaar, Roy; Abee, Tjakko

    2010-04-30

    Four strains of the Bacillus cereus group were compared for their germinant receptor composition and spore germination capacity. Phylogenetic analysis of the germinant receptor encoding operons of the enterotoxic strains B. cereus ATCC 14579 and ATCC 10987, the emetic strain AH187, and the psychrotolerant strain Bacillus weihenstephanensis KBAB4, indicated a core group of five germinant receptor operons to be present in the four strains, with each strain containing one to three additional receptors. Using quantitative PCR, induction of expression during sporulation was confirmed for all identified germinant receptor operons in these strains. Despite the large overlap in receptors, diversity in amino acid-induced germination capacity was observed, with six out of 20 amino acids, serving as germinants for spores of all four strains. Each strain showed unique features: efficient germination of strain KBAB4 spores required non-inducing amounts of inosine as the co-germinant, strain ATCC 10987 spores germinated only efficiently after heat activation. Furthermore, strain ATCC 14579 and AH187 spores germinated without heat activation or inosine, with strain ATCC 14579 spores being triggered by all amino acids except phenylalanine and strain AH187 spores being specifically triggered efficiently only by phenylalanine. Analysis of all germination data did not reveal strict linkages between specific germinants and germinant receptors. Finally, the diversity in nutrient-induced germination capacity was also reflected in the diverse germination responses of heat-activated spores of the four B. cereus strains in food matrices, such as milk, rice water and meat bouillon, indicating that amino acid composition and/or availability of inosine are important germination determinants in foods. Copyright 2010 Elsevier B.V. All rights reserved.

  3. Comparative analysis of the diversity of aerobic spore-forming bacteria in raw milk from organic and conventional dairy farms.

    Science.gov (United States)

    Coorevits, An; De Jonghe, Valerie; Vandroemme, Joachim; Reekmans, Rieka; Heyrman, Jeroen; Messens, Winy; De Vos, Paul; Heyndrickx, Marc

    2008-06-01

    Bacterial contamination of raw milk can originate from different sources: air, milking equipment, feed, soil, faeces and grass. It is hypothesized that differences in feeding and housing strategies of cows may influence the microbial quality of milk. This assumption was investigated through comparison of the aerobic spore-forming flora in milk from organic and conventional dairy farms. Laboratory pasteurized milk samples from five conventional and five organic dairy farms, sampled in late summer/autumn and in winter, were plated on a standard medium and two differential media, one screening for phospholipolytic and the other for proteolytic activity of bacteria. Almost 930 isolates were obtained of which 898 could be screened via fatty acid methyl ester analysis. Representative isolates were further analysed using 16S rRNA gene sequencing and (GTG)(5)-PCR. The majority of aerobic spore-formers in milk belonged to the genus Bacillus and showed at least 97% 16S rRNA gene sequence similarity with type strains of Bacillus licheniformis, Bacillus pumilus, Bacillus circulans, Bacillus subtilis and with type strains of species belonging to the Bacillus cereus group. About 7% of all isolates may belong to possibly new spore-forming taxa. Although the overall diversity of aerobic spore-forming bacteria in milk from organic vs. conventional dairy farms was highly similar, some differences between both were observed: (i) a relatively higher number of thermotolerant organisms in milk from conventional dairy farms compared to organic farms (41.2% vs. 25.9%), and (ii) a relatively higher number of B. cereus group organisms in milk from organic (81.3%) and Ureibacillus thermosphaericus in milk from conventional (85.7%) dairy farms. One of these differences, the higher occurrence of B. cereus group organisms in milk from organic dairy farms, may be linked to differences in housing strategy between the two types of dairy farming. However, no plausible clarification was found for

  4. Bacillus subtilis

    Science.gov (United States)

    Wang, Xiaoqing; Hu, Weiwei; Zhu, Liqi; Yang, Qian

    2017-04-28

    Intestinal epithelial cells are the targets for transmissible gastroenteritis (TGE) virus (TGEV) infection. It is urgent to develop a novel candidate against TGEV entry. Bacillus subtilis is a probiotic with excellent anti-microorganism properties and one of its secretions, surfactin, has been regarded as a versatile weapon for most plant pathogens, especially for the enveloped virus. We demonstrate for the first time that B. subtilis OKB105 and its surfactin can effectively inhibit one animal coronavirus, TGEV, entering the intestinal porcine epithelial cell line (IPEC-J2). Then, several different experiments were performed to seek the might mechanisms. The plaque assays showed that surfactant could reduce the plaque generation of TGEV in a dose-dependent manner. Meanwhile, after incubation with TGEV for 1.5 h, B. subtilis could attach TGEV particles to their surface so that the number of virus to bind to the host cells was declined. Furthermore, our data showed that the inhibition of B. subtilis was closely related to the competition with TGEV for the viral entry receptors, including epidermal growth factor receptor (EGFR) and aminopeptidase N (APN) protein. In addition, Western blotting and apoptosis analysis indicated that B. subtilis could enhance the resistance of IPEC-J2 cells by up-regulating the expression of toll-like receptor (TLR)-6 and reducing the percentage of apoptotic cells. Taken together, our results suggest that B. subtilis OKB105 and its surfactin can antagonize TGEV entry in vitro and may serve as promising new candidates for TGEV prevention. © 2017 The Author(s).

  5. Antagonistic Activities of Bacillus spp. Strains Isolated from Tidal Flat Sediment Towards Anthracnose Pathogens Colletotrichum acutatum and C. gloeosporioides in South Korea

    Directory of Open Access Journals (Sweden)

    Joon-Hee Han

    2015-06-01

    Full Text Available Anthracnose is a fungal disease caused by Colletotrichum species that is detrimental to numerous plant species. Anthracnose control with fungicides has both human health and environmental safety implications. Despite increasing public concerns, fungicide use will continue in the absence of viable alternatives. There have been relatively less efforts to search antagonistic bacteria from mudflats harboring microbial diversity. A total of 420 bacterial strains were isolated from mudflats near the western sea of South Korea. Five bacterial strains, LB01, LB14, HM03, HM17, and LB15, were characterized as having antifungal properties in the presence of C. acutatum and C. gloeosporioides. The three Bacillus atrophaeus strains, LB14, HM03, and HM17, produced large quantities of chitinase and protease enzymes, whereas the B. amyloliquefaciens strain LB01 produced protease and cellulase enzymes. Two important antagonistic traits, siderophore production and solubilization of insoluble phosphate, were observed in the three B. atrophaeus strains. Analyses of disease suppression revealed that LB14 was most effective for suppressing the incidence of anthracnose symptoms on pepper fruits. LB14 produced antagonistic compounds and suppressed conidial germination of C. acutatum and C. gloeosporioides. The results from the present study will provide a basis for developing a reliable alternative to fungicides for anthracnose control.

  6. Bacillus thuringiensis HD-1 Cry- : development of a safe, non-insecticidal simulant for Bacillus anthracis.

    Science.gov (United States)

    Bishop, A H; Robinson, C V

    2014-09-01

    A representative simulant for spores of Bacillus anthracis is needed for field testing. Bacillus thuringiensis is gaining recognition as a suitable organism. A strain that does not form the insecticidal, parasporal crystals that are characteristic of this species is a more accurate physical representative of B. anthracis spores. We developed noninsecticidal derivatives of two isolates of B. thuringiensis HD-1. Two plasmid-cured derivatives of B. thuringiensis HD-1, unable to make crystal toxins ('Cry(-) '), were isolated. These isolates and the existing Cry(-) strain, B. thuringiensis Al Hakam, were probed with PCR assays against the known insecticidal genes cry, vip and cyt. Their genomic DNA was sequenced to demonstrate a lack of insecticidal genes. This was confirmed by bioassays against a number of invertebrate species. Real-time PCR assays were developed to identify the B. thuringiensis HD-1 Cry(-) derivatives and an effective differential and selective medium was assessed. All three Cry(-) isolates are devoid of known insecticidal determinants. The B. thuringiensis HD-1 Cry(-) derivatives can easily be recovered from soil and identified by PCR with some selectivity. The B. thuringiensis HD-1 Cry(-) derivatives represent accurate, nongenetically manipulated simulants for B. anthracis with excellent human and environmental safety records. © 2014 Crown Copyright. Journal of Applied Microbiology © 2014 Society for Applied Microbiology This article is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland.

  7. Bacillus coagulans

    Science.gov (United States)

    Aulitto, Martina; Fusco, Salvatore; Bartolucci, Simonetta; Franzén, Carl Johan; Contursi, Patrizia

    2017-01-01

    The transition from a petroleum-based economy towards more sustainable bioprocesses for the production of fuels and chemicals (circular economy) is necessary to alleviate the impact of anthropic activities on the global ecosystem. Lignocellulosic biomass-derived sugars are suitable alternative feedstocks that can be fermented or biochemically converted to value-added products. An example is lactic acid, which is an essential chemical for the production of polylactic acid, a biodegradable bioplastic. However, lactic acid is still mainly produced by Lactobacillus species via fermentation of starch-containing materials, the use of which competes with the supply of food and feed. A thermophilic and cellulolytic lactic acid producer was isolated from bean processing waste and was identified as a new strain of Bacillus coagulans , named MA-13. This bacterium fermented lignocellulose-derived sugars to lactic acid at 55 °C and pH 5.5. Moreover, it was found to be a robust strain able to tolerate high concentrations of hydrolysate obtained from wheat straw pre-treated by acid-catalysed (pre-)hydrolysis and steam explosion, especially when cultivated in controlled bioreactor conditions. Indeed, unlike what was observed in microscale cultivations (complete growth inhibition at hydrolysate concentrations above 50%), B. coagulans MA-13 was able to grow and ferment in 95% hydrolysate-containing bioreactor fermentations. This bacterium was also found to secrete soluble thermophilic cellulases, which could be produced at low temperature (37 °C), still retaining an optimal operational activity at 50 °C. The above-mentioned features make B. coagulans MA-13 an appealing starting point for future development of a consolidated bioprocess for production of lactic acid from lignocellulosic biomass, after further strain development by genetic and evolutionary engineering. Its optimal temperature and pH of growth match with the operational conditions of fungal enzymes hitherto

  8. Incidence of spore-forming bacteria in unsweetened evaporated ...

    African Journals Online (AJOL)

    brand C, to 0.54x105 for brand B and 1.67x105 for brand A. The organisms isolated from the three brands were identified as Bacillus cereus, Bacillus licheniformis, Bacillus coagulans, Bacillus stearothermophilus, Bacillus subtilis and Clostridium perfringens, with B. subtilis having the highest frequency of occurrence ...

  9. Selection of Bacillus species for targeted in situ release of prebiotic galacto-rhamnogalacturonan from potato pulp in piglets

    DEFF Research Database (Denmark)

    Jers, Carsten; Strube, Mikael Lenz; Cantor, Mette D

    2017-01-01

    We have previously shown that galacto-rhamnogalacturonan fibers can be enzymatically extracted from potato pulp and that these fibers have potential for exerting a prebiotic effect in piglets. The spore-forming Bacillus species are widely used as probiotics in feed supplements for pigs. In this s...

  10. Toxicity of Tolyltriazole to Bacillus Microorganisms.

    Science.gov (United States)

    2000-03-01

    Bacillus coagulans Microbacterium lacticum Jupiter Bacillus thuringiensis Bacillus thuringiensis Bacillus cereus Bacillus Bacillus thuringiensis...TOXICITY OF TOLYLTRIAZOLE TO BACILLUS MICROORGANISMS THESIS Christopher J. Leonard, First Lieutenant, USAF AFIT/GEE/ENV/OOM-12 Approved for...AND DATES COVERED Master’s Thesis 4. TITLE AND SUBTITLE TOXICITY OF TOLYLTRIAZOLE TO BACILLUS MICROORGANISMS 6. AUTHOR(S) Christopher J

  11. Dothistroma septosporum: spore production and weather conditions

    Energy Technology Data Exchange (ETDEWEB)

    Dvorak, M.; Drapela, K.; Kankovsky, L.

    2012-11-01

    Dartmouth's septosporum, the causal agent of Dothistroma needle blight is a widespread fungus which infects more than 80 species of coniferous trees through the entire world. Spreading of the infection is strongly affected by climatic factors of each locality where it is recorded. We attempt to describe the concrete limiting climatic factors necessary for the releasing of conidia of D. septosporum and to find out the timing of its spore production within the year. For this purpose we used an automatic volumetric spore trap and an automatic meteorological station. We found that a minimum daily average temperature of 10 degree centigrade was necessary for any spore production, as well as a long period of high air humidity. The values obtained in the present study were a little bit higher than those previously published, which may arise questions about a possible changing trend of the behaviour in the development of the Dothistroma needle blight causal agent. We used autoregressive integrated moving average (ARIMA) models to predict the spore counts on the base of previous values of spore counts and dew point. For a locality from Hackerovka, the best ARIMA model was 1,0,0; and for a locality from Lanzhot, the best was 3,1,0. (Author) 19 refs.

  12. Engineering Rugged Field Assays to Detect Hazardous Chemicals Using Spore-Based Bacterial Biosensors.

    Science.gov (United States)

    Wynn, Daniel; Deo, Sapna; Daunert, Sylvia

    2017-01-01

    Bacterial whole cell-based biosensors have been genetically engineered to achieve selective and reliable detection of a wide range of hazardous chemicals. Although whole-cell biosensors demonstrate many advantages for field-based detection of target analytes, there are still some challenges that need to be addressed. Most notably, their often modest shelf life and need for special handling and storage make them challenging to use in situations where access to reagents, instrumentation, and expertise are limited. These problems can be circumvented by developing biosensors in Bacillus spores, which can be engineered to address all of these concerns. In its sporulated state, a whole cell-based biosensor has a remarkably long life span and is exceptionally resistant to environmental insult. When these spores are germinated for use in analytical techniques, they show no loss in performance, even after long periods of storage under harsh conditions. In this chapter, we will discuss the development and use of whole cell-based sensors, their adaptation to spore-based biosensors, their current applications, and future directions in the field. © 2017 Elsevier Inc. All rights reserved.

  13. Isolation and Characterization of Cryptococcus neoformans Spores Reveal a Critical Role for Capsule Biosynthesis Genes in Spore Biogenesis▿

    Science.gov (United States)

    Botts, Michael R.; Giles, Steven S.; Gates, Marcellene A.; Kozel, Thomas R.; Hull, Christina M.

    2009-01-01

    Spores are essential particles for the survival of many organisms, both prokaryotic and eukaryotic. Among the eukaryotes, fungi have developed spores with superior resistance and dispersal properties. For the human fungal pathogens, however, relatively little is known about the role that spores play in dispersal and infection. Here we present the purification and characterization of spores from the environmental fungus Cryptococcus neoformans. For the first time, we purified spores to homogeneity and assessed their morphological, stress resistance, and surface properties. We found that spores are morphologically distinct from yeast cells and are covered with a thick spore coat. Spores are also more resistant to environmental stresses than yeast cells and display a spore-specific configuration of polysaccharides on their surfaces. Surprisingly, we found that the surface of the spore reacts with antibodies to the polysaccharide glucuronoxylomannan, the most abundant component of the polysaccharide capsule required for C. neoformans virulence. We explored the role of capsule polysaccharide in spore development by assessing spore formation in a series of acapsular strains and determined that capsule biosynthesis genes are required for proper sexual development and normal spore formation. Our findings suggest that C. neoformans spores may have an adapted cell surface that facilitates persistence in harsh environments and ultimately allows them to infect mammalian hosts. PMID:19181873

  14. [Bioconversion of sewage sludge to biopesticide by Bacillus thuringiensis].

    Science.gov (United States)

    Chang, Ming; Zhou, Shun-gui; Lu, Na; Ni, Jin-ren

    2006-07-01

    Feasibility of bioconversion of sewage sludge to biopesticide by Bacillus thuringiensis was studied using sewage sludge as a raw material. The fermentation was also compared with conventional medium. Results showed that without any pretreatment, the nutrients contained in sewage sludge were almost sufficient for Bacillus thuringiensis growth, even with a rapid multiplicational rate. Higher viable cells and viable spores values were obtained earlier at 24 h, with 9.48 x 10(8) CFU x mL(-1) and 8.51 x 10(8) CFU x mL(-1) respectively, which was 12 hours earlier and nearly 20 percent higher than conventional medium. SEM of 36 h samples gave a clear phenomenon that the metabolizability in sludge was much faster with spores and crystals spreading around. The crystals in sludge seemed rather bigger and more regular. Also a better crystal protein yield of 2.80 mg x mL(-1) was observed in sludge medium compared to conventional medium at the end of fermentation. Sludge fermentation for Bacillus thuringiensis reduces the producing cost, and gives better fermentation capabilities. It's expected to be a new method for sludge disposal.

  15. Selection of Bacillus species for targeted in situ release of prebiotic galacto-rhamnogalacturonan from potato pulp in piglets.

    Science.gov (United States)

    Jers, Carsten; Strube, Mikael L; Cantor, Mette D; Nielsen, Bea K K; Sørensen, Ole B; Boye, Mette; Meyer, Anne S

    2017-05-01

    We have previously shown that galacto-rhamnogalacturonan fibers can be enzymatically extracted from potato pulp and that these fibers have potential for exerting a prebiotic effect in piglets. The spore-forming Bacillus species are widely used as probiotics in feed supplements for pigs. In this study, we evaluated the option for further functionalizing Bacillus feed supplements by selecting strains possessing the enzymes required for extraction of the potentially prebiotic fibers. We established that it would require production and secretion of pectin lyase and/or polygalacturonase but no or limited secretion of galactanase and β-galactosidase. By screening a library of 158 Bacillus species isolated from feces and soil, we demonstrated that especially strains of Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus mojavensis have the necessary enzyme profile and thus the capability to degrade polygalacturonan. Using an in vitro porcine gastrointestinal model system, we revealed that specifically strains of B. mojavensis were able to efficiently release galacto-rhamnogalacturonan from potato pulp under simulated gastrointestinal conditions. The work thus demonstrated the feasibility of producing prebiotic fibers via a feed containing Bacillus spores and potato pulp and identified candidates for future in vivo evaluation in piglets.

  16. Handling technique of spore-forming bacteria in radiation sterilization. 2. Determination of numbers and radiation resistance of spores

    International Nuclear Information System (INIS)

    Koshikawa, Tomihiko

    1994-01-01

    Stepwise ten-fold dilution of bacterial solution is required in the determination of bacterial spores. For this, the selection of diluted solution is important according to the purpose of experiment. First, the preparation of suspension of bacterial spores and selection of diluted solution are presented. Then, a method for determining the number of bacterial spores in materials is outlined in terms of dilution methods of bacterial solution (shaking and homogenization) and application method of diluted solution to the plating medium. Finally, a method for determining radiation resistance of spore-forming bacteria is explained according to the measurement conditions (suspension of bacterial spores and filters applied with bacterial spores). (N.K.)

  17. Handling technique of spore-forming bacteria in radiation sterilization. 2. Determination of numbers and radiation resistance of spores

    Energy Technology Data Exchange (ETDEWEB)

    Koshikawa, Tomihiko [Japan Radioisotope Association, Shiga (Japan). Koka Laboratory

    1994-12-01

    Stepwise ten-fold dilution of bacterial solution is required in the determination of bacterial spores. For this, the selection of diluted solution is important according to the purpose of experiment. First, the preparation of suspension of bacterial spores and selection of diluted solution are presented. Then, a method for determining the number of bacterial spores in materials is outlined in terms of dilution methods of bacterial solution (shaking and homogenization) and application method of diluted solution to the plating medium. Finally, a method for determining radiation resistance of spore-forming bacteria is explained according to the measurement conditions (suspension of bacterial spores and filters applied with bacterial spores). (N.K.).

  18. Combined high pressure and thermal processing on inactivation of type A and proteolytic type B spores of Clostridium botulinum.

    Science.gov (United States)

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

    2013-08-01

    The aim of this study was to determine the resistance of multiple strains of Clostridium botulinum type A and proteolytic type B spores exposed to combined high pressure and thermal processing and compare their resistance with Clostridium sporogenes PA3679 and Bacillus amyloliquefaciens TMW-2.479-Fad-82 spores. The resistance of spores suspended in N-(2acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7.0) was determined at a process temperature of 105°C, with high pressures of 600, 700, and 750 MPa by using a laboratory-scale pressure test system. No surviving spores of the proteolytic B strains were detected after processing at 105°C and 700 MPa for 6 min. A . 7-log reduction of B. amyloliquefaciens spores was observed when processed for 4 min at 105°C and 700 MPa. D-values at 105°C and 700 MPa for type A strains ranged from 0.57 to 2.28 min. C. sporogenes PA3679 had a D-value of 1.48 min at 105°C and 700 MPa. Spores of the six type A strains with high D-values along with C. sporogenes PA3679 and B. amyloliquefaciens were further evaluated for their pressure resistance at pressures 600 and 750 MPa at 105°C. As the process pressure increased from 600 to 750 MPa at 105°C, D-values of some C. botulinum strains and C. sporogenes PA3679 spores decreased (i.e., 69-A, 1.91 to 1.33 min and PA3679, 2.35 to 1.29 min). Some C. botulinum type A strains were more resistant than C. sporogenes PA3679 and B. amyloliquefaciens to combined high pressure and heat, based on D-values determined at 105°C. Pulsed-field gel electrophoresis (PFGE) was also performed to establish whether strains with a similar restriction banding pattern also exhibited similar D-values. However, no correlation between the genomic background of a strain and its resistance to high pressure processing was observed, based on PFGE analysis. Spores of proteolytic type B strains of C. botulinum were less resistant to combined high pressure and heat (700 MPa and 105°C) treatment when

  19. Advances in developing Bacillus thuringiensis-based insecticde formulations Avances en el desarrollo de formulaciones insecticidas a base de Bacillus thuringiensis

    OpenAIRE

    Rosas-García Ninfa María

    2008-01-01

    Developing Bacillus thuringiensis-based formulations is an old technology which has been revived during recent decades. The spore-crystal complex (being the main ingredient in these preparations) has been the main objective of this research, involving the search for new or improved strains. The type of materials used included a wide variety of completely biodegradable ingredients which could have been leaves, stems or fruit which when dried and ground could serve as feeding stimulants, as wel...

  20. Effect of Essential Oils on Germination and Growth of Some Pathogenic and Spoilage Spore-Forming Bacteria.

    Science.gov (United States)

    Voundi, Stève Olugu; Nyegue, Maximilienne; Lazar, Iuliana; Raducanu, Dumitra; Ndoye, Florentine Foe; Marius, Stamate; Etoa, François-Xavier

    2015-06-01

    The use of essential oils as a food preservative has increased due to their capacity to inhibit vegetative growth of some bacteria. However, only limited data are available on their effect on bacterial spores. The aim of the present study was to evaluate the effect of some essential oils on the growth and germination of three Bacillus species and Geobacillus stearothermophilus. Essential oils were chemically analyzed using gas chromatography and gas chromatography coupled to mass spectrometry. The minimal inhibitory and bactericidal concentrations of vegetative growth and spore germination were assessed using the macrodilution method. Germination inhibitory effect of treated spores with essential oils was evaluated on solid medium, while kinetic growth was followed using spectrophotometry in the presence of essential oils. Essential oil from Drypetes gossweileri mainly composed of benzyl isothiocyanate (86.7%) was the most potent, with minimal inhibitory concentrations ranging from 0.0048 to 0.0097 mg/mL on vegetative cells and 0.001 to 0.002 mg/mL on spore germination. Furthermore, essential oil from D. gossweileri reduced 50% of spore germination after treatment at 1.25 mg/mL, and its combination with other oils improved both bacteriostatic and bactericidal activities with additive or synergistic effects. Concerning the other essential oils, the minimal inhibitory concentration ranged from 5 to 0.63 mg/mL on vegetative growth and from 0.75 to 0.09 mg/mL on the germination of spores. Spectrophotometric evaluation showed an inhibitory effect of essential oils on both germination and outgrowth. From these results, it is concluded that some of the essential oils tested might be a valuable tool for bacteriological control in food industries. Therefore, further research regarding their use as food preservatives should be carried out.

  1. Influence of Heat Shock Temperatures and Fast Freezing on Viability of Probiotic Sporeformers and the Issue of Spore Plate Count Versus True Numbers

    Directory of Open Access Journals (Sweden)

    Mojtaba Jafari

    2016-02-01

    Full Text Available Background and Objectives: The purpose of the present study was to investigate effects of various heat shock conditions and fast freezing and subsequent thawing on the viability and recovery of Bacillus coagulans and Bacillus subtilis as probiotic sporeformers, and also to compare spore plate and microscopic counts. Materials and Methods: After preparing the final suspensions of B. coagulans and Bacillus subtilis subsp. Natto spores, they were spread-plated before and after fast freezing treatment (-70°C for about 1 min. Heat shock treatments of the spores were carried out at 68oC for 15, 20, and 30 min as well as at 80oC for 10 and 15 min. Concentrations of the examined probiotic sporeformers were determined simultaneously by plate enumerations and microscopically determined counts. Student’s t-test and one-way analysis of variance (ANOVA of SPSS were used for statistical analysis of the data. Analysis of DoE results was carried out using Minitab. Results: The results presented here show that the highest recovery rates for B. coagulans (14.75 log CFU/mL and B. subtilis spores (14.80 log CFU/mL were under a heat shock condition of 68°C for 20 min in nutrient agar (p<0.05. In addition, the survival rates of B. coagulans and B. subtilis spores under the fast freezing and subsequent thawing condition were about 90% and 88%, respectively. Plate counts differed significantly from counts determined microscopically, with differences of almost 0.5 and 0.8 log for B. coagulans and B. subtilis spores, respectively (p<0.05. In addition, DoE results of the study revealed that both factors of spore count method and only freezing factor in fast freezing treatment have a significant effect on concentrations of the spores examined (p<0.05. Conclusions: Heat shock conditions, freezing and subsequent thawing circumstances, and plate counts or enumerations determined microscopically have significant influences on the viability of probiotic sporeformers and

  2. Fifth international fungus spore conference. [Abstracts]: Final technical report

    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.

  3. Genome Sequence of Bacillus endophyticus and Analysis of Its Companion Mechanism in the Ketogulonigenium vulgare-Bacillus Strain Consortium.

    Directory of Open Access Journals (Sweden)

    Nan Jia

    Full Text Available Baci