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Sample records for anthracis bacteriophage-mediated ecological

  1. The secret life of the anthrax agent Bacillus anthracis: bacteriophage-mediated ecological adaptations.

    Science.gov (United States)

    Schuch, Raymond; Fischetti, Vincent A

    2009-08-12

    Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities.

  2. The secret life of the anthrax agent Bacillus anthracis: bacteriophage-mediated ecological adaptations.

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    Raymond Schuch

    Full Text Available Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities.

  3. Redefining the Australian Anthrax Belt: Modeling the Ecological Niche and Predicting the Geographic Distribution of Bacillus anthracis.

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    Alassane S Barro

    2016-06-01

    Full Text Available The ecology and distribution of B. anthracis in Australia is not well understood, despite the continued occurrence of anthrax outbreaks in the eastern states of the country. Efforts to estimate the spatial extent of the risk of disease have been limited to a qualitative definition of an anthrax belt extending from southeast Queensland through the centre of New South Wales and into northern Victoria. This definition of the anthrax belt does not consider the role of environmental conditions in the distribution of B. anthracis. Here, we used the genetic algorithm for rule-set prediction model system (GARP, historical anthrax outbreaks and environmental data to model the ecological niche of B. anthracis and predict its potential geographic distribution in Australia. Our models reveal the niche of B. anthracis in Australia is characterized by a narrow range of ecological conditions concentrated in two disjunct corridors. The most dominant corridor, used to redefine a new anthrax belt, parallels the Eastern Highlands and runs from north Victoria to central east Queensland through the centre of New South Wales. This study has redefined the anthrax belt in eastern Australia and provides insights about the ecological factors that limit the distribution of B. anthracis at the continental scale for Australia. The geographic distributions identified can help inform anthrax surveillance strategies by public and veterinary health agencies.

  4. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

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    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis.

  5. Human Neutrophils Kill Bacillus anthracis.

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    2005-11-01

    Full Text Available Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

  6. Human neutrophils kill Bacillus anthracis.

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    Anne Mayer-Scholl

    2005-11-01

    Full Text Available Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

  7. Human neutrophils kill Bacillus anthracis.

    Science.gov (United States)

    Mayer-Scholl, Anne; Hurwitz, Robert; Brinkmann, Volker; Schmid, Monika; Jungblut, Peter; Weinrauch, Yvette; Zychlinsky, Arturo

    2005-11-01

    Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

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

    OpenAIRE

    1983-01-01

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

  9. Synthesis of lipoteichoic acids in Bacillus anthracis.

    Science.gov (United States)

    Garufi, Gabriella; Hendrickx, Antoni P; Beeri, Karen; Kern, Justin W; Sharma, Anshika; Richter, Stefan G; Schneewind, Olaf; Missiakas, Dominique

    2012-08-01

    Lipoteichoic acid (LTA), a glycerol phosphate polymer, is a component of the envelope of Gram-positive bacteria that has hitherto not been identified in Bacillus anthracis, the causative agent of anthrax. LTA synthesis in Staphylococcus aureus and other microbes is catalyzed by the product of the ltaS gene, a membrane protein that polymerizes polyglycerol phosphate from phosphatidyl glycerol. Here we identified four ltaS homologues, designated ltaS1 to -4, in the genome of Bacillus anthracis. Polyglycerol phosphate-specific monoclonal antibodies were used to detect LTA in the envelope of B. anthracis strain Sterne (pXO1(+) pXO2(-)) vegetative forms. B. anthracis mutants lacking ltaS1, ltaS2, ltaS3, or ltaS4 did not display defects in growth or LTA synthesis. In contrast, B. anthracis strains lacking both ltaS1 and ltaS2 were unable to synthesize LTA and exhibited reduced viability, altered envelope morphology, aberrant separation of vegetative forms, and decreased sporulation efficiency. Expression of ltaS1 or ltaS2 alone in B. anthracis as well as in other microbes was sufficient for polyglycerol phosphate synthesis. Thus, similar to S. aureus, B. anthracis employs LtaS enzymes to synthesize LTA, an envelope component that promotes bacterial growth and cell division.

  10. Bacillus anthracis factors for phagosomal escape.

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    Tonello, Fiorella; Zornetta, Irene

    2012-07-01

    The mechanism of phagosome escape by intracellular pathogens is an important step in the infectious cycle. During the establishment of anthrax, Bacillus anthracis undergoes a transient intracellular phase in which spores are engulfed by local phagocytes. Spores germinate inside phagosomes and grow to vegetative bacilli, which emerge from their resident intracellular compartments, replicate and eventually exit from the plasma membrane. During germination, B. anthracis secretes multiple factors that can help its resistance to the phagocytes. Here the possible role of B. anthracis toxins, phospholipases, antioxidant enzymes and capsules in the phagosomal escape and survival, is analyzed and compared with that of factors of other microbial pathogens involved in the same type of process.

  11. Bacillus anthracis Diversity and Geographic Potential across Nigeria, Cameroon and Chad: Further Support of a Novel West African Lineage.

    Science.gov (United States)

    Blackburn, Jason K; Odugbo, Moses Ode; Van Ert, Matthew; O'Shea, Bob; Mullins, Jocelyn; Perreten, Vincent; Perrenten, Vincent; Maho, Angaya; Hugh-Jones, Martin; Hadfield, Ted

    2015-01-01

    Zoonoses, diseases affecting both humans and animals, can exert tremendous pressures on human and veterinary health systems, particularly in resource limited countries. Anthrax is one such zoonosis of concern and is a disease requiring greater public health attention in Nigeria. Here we describe the genetic diversity of Bacillus anthracis in Nigeria and compare it to Chad, Cameroon and a broader global dataset based on the multiple locus variable number tandem repeat (MLVA-25) genetic typing system. Nigerian B. anthracis isolates had identical MLVA genotypes and could only be resolved by measuring highly mutable single nucleotide repeats (SNRs). The Nigerian MLVA genotype was identical or highly genetically similar to those in the neighboring countries, confirming the strains belong to this unique West African lineage. Interestingly, sequence data from a Nigerian isolate shares the anthrose deficient genotypes previously described for strains in this region, which may be associated with vaccine evasion. Strains in this study were isolated over six decades, indicating a high level of temporal strain stability regionally. Ecological niche models were used to predict the geographic distribution of the pathogen for all three countries. We describe a west-east habitat corridor through northern Nigeria extending into Chad and Cameroon. Ecological niche models and genetic results show B. anthracis to be ecologically established in Nigeria. These findings expand our understanding of the global B. anthracis population structure and can guide regional anthrax surveillance and control planning.

  12. Bacillus anthracis Diversity and Geographic Potential across Nigeria, Cameroon and Chad: Further Support of a Novel West African Lineage.

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    Jason K Blackburn

    Full Text Available Zoonoses, diseases affecting both humans and animals, can exert tremendous pressures on human and veterinary health systems, particularly in resource limited countries. Anthrax is one such zoonosis of concern and is a disease requiring greater public health attention in Nigeria. Here we describe the genetic diversity of Bacillus anthracis in Nigeria and compare it to Chad, Cameroon and a broader global dataset based on the multiple locus variable number tandem repeat (MLVA-25 genetic typing system. Nigerian B. anthracis isolates had identical MLVA genotypes and could only be resolved by measuring highly mutable single nucleotide repeats (SNRs. The Nigerian MLVA genotype was identical or highly genetically similar to those in the neighboring countries, confirming the strains belong to this unique West African lineage. Interestingly, sequence data from a Nigerian isolate shares the anthrose deficient genotypes previously described for strains in this region, which may be associated with vaccine evasion. Strains in this study were isolated over six decades, indicating a high level of temporal strain stability regionally. Ecological niche models were used to predict the geographic distribution of the pathogen for all three countries. We describe a west-east habitat corridor through northern Nigeria extending into Chad and Cameroon. Ecological niche models and genetic results show B. anthracis to be ecologically established in Nigeria. These findings expand our understanding of the global B. anthracis population structure and can guide regional anthrax surveillance and control planning.

  13. Specific identification of Bacillus anthracis strains

    Science.gov (United States)

    Krishnamurthy, Thaiya; Deshpande, Samir; Hewel, Johannes; Liu, Hongbin; Wick, Charles H.; Yates, John R., III

    2007-01-01

    Accurate identification of human pathogens is the initial vital step in treating the civilian terrorism victims and military personnel afflicted in biological threat situations. We have applied a powerful multi-dimensional protein identification technology (MudPIT) along with newly generated software termed Profiler to identify the sequences of specific proteins observed for few strains of Bacillus anthracis, a human pathogen. Software termed Profiler was created to initially screen the MudPIT data of B. anthracis strains and establish the observed proteins specific for its strains. A database was also generated using Profiler containing marker proteins of B. anthracis and its strains, which in turn could be used for detecting the organism and its corresponding strains in samples. Analysis of the unknowns by our methodology, combining MudPIT and Profiler, led to the accurate identification of the anthracis strains present in samples. Thus, a new approach for the identification of B. anthracis strains in unknown samples, based on the molecular mass and sequences of marker proteins, has been ascertained.

  14. Bacillus anthracis diversity in Kruger National Park.

    Science.gov (United States)

    Smith, K L; DeVos, V; Bryden, H; Price, L B; Hugh-Jones, M E; Keim, P

    2000-10-01

    The Kruger National Park (KNP), South Africa, has a recorded history of periodic anthrax epidemics causing widespread disease among wild animals. Bacillus anthracis is the causative agent of anthrax, a disease primarily affecting ungulate herbivores. Worldwide there is little diversity among B. anthracis isolates, but examination of variable-number tandem repeat (VNTR) loci has identified six major clones, with the most dissimilar types split into the A and B branches. Both the A and B types are found in southern Africa, giving this region the greatest genetic diversity of B. anthracis worldwide. Consequently, southern Africa has been hypothesized to be the geographic origin of B. anthracis. In this study, we identify the genotypic types of 98 KNP B. anthracis isolates using multiple-locus VNTR analysis. Two major types are evident, the A branch and the B branch. The spatial and temporal distribution of the different genotypes indicates that anthrax epidemic foci are independent, though correlated through environmental cues. Kruger B isolates were found on significantly higher-calcium and higher-pH soils than were Kruger type A. This relationship between genotype and soil chemistry may be due to adaptive differences among divergent anthrax strains. While this association may be simply fortuitous, adaptation of A types to diverse environmental conditions is consistent with their greater geographic dispersal and genetic dissimilarity.

  15. Bacillus anthracis Bioterrorism Incident, Kameido, Tokyo, 1993

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    Keim, Paul; Kaufmann, Arnold F.; Keys, Christine; Smith, Kimothy L.; Taniguchi, Kiyosu; Inouye, Sakae; Kurata, Takeshi

    2004-01-01

    In July 1993, a liquid suspension of Bacillus anthracis was aerosolized from the roof of an eight-story building in Kameido, Tokyo, Japan, by the religious group Aum Shinrikyo. During 1999 to 2001, microbiologic tests were conducted on a liquid environmental sample originally collected during the 1993 incident. Nonencapsulated isolates of B. anthracis were cultured from the liquid. Multiple-locus, variable-number tandem repeat analysis found all isolates to be identical to a strain used in Japan to vaccinate animals against anthrax, which was consistent with the Aum Shinrikyo members’ testimony about the strain source. In 1999, a retrospective case-detection survey was conducted to identify potential human anthrax cases associated with the incident, but none were found. The use of an attenuated B. anthracis strain, low spore concentrations, ineffective dispersal, a clogged spray device, and inactivation of the spores by sunlight are all likely contributing factors to the lack of human cases. PMID:15112666

  16. Methyl Iodide Fumigation of Bacillus anthracis Spores.

    Science.gov (United States)

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

    2015-09-01

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

  17. Characterization of Bacillus anthracis persistence in vivo.

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    Sarah A Jenkins

    Full Text Available Pulmonary exposure to Bacillus anthracis spores initiates inhalational anthrax, a life-threatening infection. It is known that dormant spores can be recovered from the lungs of infected animals months after the initial spore exposure. Consequently, a 60-day course antibiotic treatment is recommended for exposed individuals. However, there has been little information regarding details or mechanisms of spore persistence in vivo. In this study, we investigated spore persistence in a mouse model. The results indicated that weeks after intranasal inoculation with B. anthracis spores, substantial amounts of spores could be recovered from the mouse lung. Moreover, spores of B. anthracis were significantly better at persisting in the lung than spores of a non-pathogenic Bacillus subtilis strain. The majority of B. anthracis spores in the lung were tightly associated with the lung tissue, as they could not be readily removed by lavage. Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium. Confocal analysis indicated that some of the spores were inside epithelial cells. This was further confirmed by differential immunofluorescence staining of lung cells harvested from the infected lungs, suggesting that association with lung epithelial cells may provide an advantage to spore persistence in the lung. There was no or very mild inflammation in the infected lungs. Furthermore, spores were present in the lung tissue as single spores rather than in clusters. We also showed that the anthrax toxins did not play a role in persistence. Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.

  18. Distribution and molecular evolution of bacillus anthracis genotypes in Namibia.

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    Wolfgang Beyer

    Full Text Available The recent development of genetic markers for Bacillus anthracis has made it possible to monitor the spread and distribution of this pathogen during and between anthrax outbreaks. In Namibia, anthrax outbreaks occur annually in the Etosha National Park (ENP and on private game and livestock farms. We genotyped 384 B. anthracis isolates collected between 1983-2010 to identify the possible epidemiological correlations of anthrax outbreaks within and outside the ENP and to analyze genetic relationships between isolates from domestic and wild animals. The isolates came from 20 animal species and from the environment and were genotyped using a 31-marker multi-locus-VNTR-analysis (MLVA and, in part, by twelve single nucleotide polymorphism (SNP markers and four single nucleotide repeat (SNR markers. A total of 37 genotypes (GT were identified by MLVA, belonging to four SNP-groups. All GTs belonged to the A-branch in the cluster- and SNP-analyses. Thirteen GTs were found only outside the ENP, 18 only within the ENP and 6 both inside and outside. Genetic distances between isolates increased with increasing time between isolations. However, genetic distance between isolates at the beginning and end of the study period was relatively small, indicating that while the majority of GTs were only found sporadically, three genetically close GTs, accounting for more than four fifths of all the ENP isolates, appeared dominant throughout the study period. Genetic distances among isolates were significantly greater for isolates from different host species, but this effect was small, suggesting that while species-specific ecological factors may affect exposure processes, transmission cycles in different host species are still highly interrelated. The MLVA data were further used to establish a model of the probable evolution of GTs within the endemic region of the ENP. SNR-analysis was helpful in correlating an isolate with its source but did not elucidate

  19. Morphogenesis of the Bacillus anthracis Spore

    Science.gov (United States)

    2007-02-01

    Fritsch, and T. Maniatis . 1989. Molecular cloning : a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 72. Santo...Sciences, Illinois Institute of Technology, Chicago, Illinois 606163; Department of Molecular , Cellular, and Developmental Biology, University of...collection E. coli DH5 Cloning host Laboratory collection BL21(DE3) Overproduction host Novagen GM1684 dam; for transformation of B. anthracis T. Koehler

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

    Science.gov (United States)

    2007-11-02

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

  1. Interactions between Bacillus anthracis and plants may promote anthrax transmission.

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    Holly H Ganz

    2014-06-01

    Full Text Available Environmental reservoirs are essential in the maintenance and transmission of anthrax but are poorly characterized. The anthrax agent, Bacillus anthracis was long considered an obligate pathogen that is dormant and passively transmitted in the environment. However, a growing number of laboratory studies indicate that, like some of its close relatives, B. anthracis has some activity outside of its vertebrate hosts. Here we show in the field that B. anthracis has significant interactions with a grass that could promote anthrax spore transmission to grazing hosts. Using a local, virulent strain of B. anthracis, we performed a field experiment in an enclosure within a grassland savanna. We found that B. anthracis increased the rate of establishment of a native grass (Enneapogon desvauxii by 50% and that grass seeds exposed to blood reached heights that were 45% taller than controls. Further we detected significant effects of E. desvauxii, B. anthracis, and their interaction on soil bacterial taxa richness and community composition. We did not find any evidence for multiplication or increased longevity of B. anthracis in bulk soil associated with grass compared to controls. Instead interactions between B. anthracis and plants may result in increased host grazing and subsequently increased transmission to hosts.

  2. Genetic Characterization of Bacillus anthracis 17 JB strain

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    Sakineh Seyed-Mohamadi

    2015-11-01

    Full Text Available Background and Objectives: Bacillus anthracis is one of the most homogenous bacteria ever described. Bacillus anthracis 17JB is a laboratory strain. It is broadly used as a challenge strain in guinea pigs for potency test of anthrax vaccine.Material and Methods: This work describes genetic characterization of B. anthracis 17 JB strain using the SNPs and MLVA genotyping.Results and Conclusion: In SNPs typing, the originally French 17JB strain represented the A. Br. 008/009 subgroup. In Levy's genotyping method, 843, 451 and 864 bp long fragments were identified at AA03, AJ03 and AA07 loci, respectively. In the vaccine manufacturer perspective these findings are much valuable on their own account, but similar research is required to extend molecular knowledge of B. anthracis epidemiology in Persia.Keywords: Bacillus anthracis 17JB, Genetic characterization, SNPs typing

  3. 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.

  4. Inactivation of Bacillus Anthracis Spores Using Carbon Nanotubes

    Science.gov (United States)

    2014-10-30

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

  5. Cortex Peptidoglycan Lytic Activity in Germinating Bacillus anthracis Spores▿

    OpenAIRE

    2008-01-01

    Bacterial endospore dormancy and resistance properties depend on the relative dehydration of the spore core, which is maintained by the spore membrane and its surrounding cortex peptidoglycan wall. During spore germination, the cortex peptidoglycan is rapidly hydrolyzed by lytic enzymes packaged into the dormant spore. The peptidoglycan structures in both dormant and germinating Bacillus anthracis Sterne spores were analyzed. The B. anthracis dormant spore peptidoglycan was similar to that fo...

  6. Phosphate starvation enhances the pathogenesis of Bacillus anthracis.

    Science.gov (United States)

    Aggarwal, Somya; Somani, Vikas Kumar; Bhatnagar, Rakesh

    2015-09-01

    Identifying the factors responsible for survival and virulence of Bacillus anthracis within the host is prerequisite for the development of therapeutics against anthrax. Host provides several stresses as well as many advantages to the invading pathogen. Inorganic phosphate (Pi) starvation within the host has been considered as one of the major contributing factors in the establishment of infection by pathogenic microorganisms. Here, we report for the first time that Pi fluctuation encountered by B. anthracis at different stages of its life cycle within the host, contributes significantly in its pathogenesis. In this study, Pi starvation was found to hasten the onset of infection cycle by promoting spore germination. After germination, it was found to impede cell growth. In addition, phosphate starved bacilli showed more antibiotic tolerance. Interestingly, phosphate starvation enhanced the pathogenicity of B. anthracis by augmenting its invasiveness in macrophages in vitro. B. anthracis grown under phosphate starvation were also found to be more efficient in establishing lethal infections in mouse model as well. Phosphate starvation increased B. anthracis virulence by promoting the secretion of primary virulence factors like protective antigen (PA), lethal factor (LF) and edema factor (EF). Thus, this study affirms that besides other host mediated factors, phosphate limitation may also contribute B. anthracis for successfully establishing itself within the host. This study is a step forward in delineating its pathophysiology that might help in understanding the pathogenesis of anthrax.

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

    OpenAIRE

    2016-01-01

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

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

    Science.gov (United States)

    2008-03-01

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

  9. Wide Area Recovery and Resiliency Program (WARRP) Interim Clearance Strategy for Environments Contaminated with Bacillus anthracis

    Science.gov (United States)

    2012-07-01

    Interim Clearance Strategy for Environments Contaminated with Bacillus anthracis July 2012...WARRP) Interim Clearance Strategy for Environments Contaminated with Bacillus anthracis 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT...contains color images. 14. ABSTRACT If a Bacillus anthracis incident occurs in the United States or within its territories, the public health and

  10. Novel giant siphovirus from Bacillus anthracis features unusual genome characteristics.

    Directory of Open Access Journals (Sweden)

    Holly H Ganz

    Full Text Available Here we present vB_BanS-Tsamsa, a novel temperate phage isolated from Bacillus anthracis, the agent responsible for anthrax infections in wildlife, livestock and humans. Tsamsa phage is a giant siphovirus (order Caudovirales, featuring a long, flexible and non-contractile tail of 440 nm (not including baseplate structure and an isometric head of 82 nm in diameter. We induced Tsamsa phage in samples from two different carcass sites in Etosha National Park, Namibia. The Tsamsa phage genome is the largest sequenced Bacillus siphovirus, containing 168,876 bp and 272 ORFs. The genome features an integrase/recombinase enzyme, indicative of a temperate lifestyle. Among bacterial strains tested, the phage infected only certain members of the Bacillus cereus sensu lato group (B. anthracis, B. cereus and B. thuringiensis and exhibited moderate specificity for B. anthracis. Tsamsa lysed seven out of 25 B. cereus strains, two out of five B. thuringiensis strains and six out of seven B. anthracis strains tested. It did not lyse B. anthracis PAK-1, an atypical strain that is also resistant to both gamma phage and cherry phage. The Tsamsa endolysin features a broader lytic spectrum than the phage host range, indicating possible use of the enzyme in Bacillus biocontrol.

  11. Novel giant siphovirus from Bacillus anthracis features unusual genome characteristics.

    Science.gov (United States)

    Ganz, Holly H; Law, Christina; Schmuki, Martina; Eichenseher, Fritz; Calendar, Richard; Loessner, Martin J; Getz, Wayne M; Korlach, Jonas; Beyer, Wolfgang; Klumpp, Jochen

    2014-01-01

    Here we present vB_BanS-Tsamsa, a novel temperate phage isolated from Bacillus anthracis, the agent responsible for anthrax infections in wildlife, livestock and humans. Tsamsa phage is a giant siphovirus (order Caudovirales), featuring a long, flexible and non-contractile tail of 440 nm (not including baseplate structure) and an isometric head of 82 nm in diameter. We induced Tsamsa phage in samples from two different carcass sites in Etosha National Park, Namibia. The Tsamsa phage genome is the largest sequenced Bacillus siphovirus, containing 168,876 bp and 272 ORFs. The genome features an integrase/recombinase enzyme, indicative of a temperate lifestyle. Among bacterial strains tested, the phage infected only certain members of the Bacillus cereus sensu lato group (B. anthracis, B. cereus and B. thuringiensis) and exhibited moderate specificity for B. anthracis. Tsamsa lysed seven out of 25 B. cereus strains, two out of five B. thuringiensis strains and six out of seven B. anthracis strains tested. It did not lyse B. anthracis PAK-1, an atypical strain that is also resistant to both gamma phage and cherry phage. The Tsamsa endolysin features a broader lytic spectrum than the phage host range, indicating possible use of the enzyme in Bacillus biocontrol.

  12. Bacillus anthracis lethal toxin reduces human alveolar epithelial barrier function.

    Science.gov (United States)

    Langer, Marybeth; Duggan, Elizabeth Stewart; Booth, John Leland; Patel, Vineet Indrajit; Zander, Ryan A; Silasi-Mansat, Robert; Ramani, Vijay; Veres, Tibor Zoltan; Prenzler, Frauke; Sewald, Katherina; Williams, Daniel M; Coggeshall, Kenneth Mark; Awasthi, Shanjana; Lupu, Florea; Burian, Dennis; Ballard, Jimmy Dale; Braun, Armin; Metcalf, Jordan Patrick

    2012-12-01

    The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness.

  13. Bacillus anthracis Factors for Phagosomal Escape

    Directory of Open Access Journals (Sweden)

    Irene Zornetta

    2012-07-01

    Full Text Available The mechanism of phagosome escape by intracellular pathogens is an important step in the infectious cycle. During the establishment of anthrax, Bacillus anthracis undergoes a transient intracellular phase in which spores are engulfed by local phagocytes. Spores germinate inside phagosomes and grow to vegetative bacilli, which emerge from their resident intracellular compartments, replicate and eventually exit from the plasma membrane. During germination, B. anthracis secretes multiple factors that can help its resistance to the phagocytes. Here the possible role of B. anthracis toxins, phospholipases, antioxidant enzymes and capsules in the phagosomal escape and survival, is analyzed and compared with that of factors of other microbial pathogens involved in the same type of process.

  14. Genetic variation and linkage disequilibrium in Bacillus anthracis.

    Science.gov (United States)

    Zwick, Michael E; Thomason, Maureen Kiley; Chen, Peter E; Johnson, Henry R; Sozhamannan, Shanmuga; Mateczun, Alfred; Read, Timothy D

    2011-01-01

    We performed whole-genome amplification followed by hybridization of custom-designed resequencing arrays to resequence 303 kb of genomic sequence from a worldwide panel of 39 Bacillus anthracis strains. We used an efficient algorithm contained within a custom software program, UniqueMER, to identify and mask repetitive sequences on the resequencing array to reduce false-positive identification of genetic variation, which can arise from cross-hybridization. We discovered a total of 240 single nucleotide variants (SNVs) and showed that B. anthracis strains have an average of 2.25 differences per 10,000 bases in the region we resequenced. Common SNVs in this region are found to be in complete linkage disequilibrium. These patterns of variation suggest there has been little if any historical recombination among B. anthracis strains since the origin of the pathogen. This pattern of common genetic variation suggests a framework for recognizing new or genetically engineered strains.

  15. Use of long-range repetitive element polymorphism-PCR to differentiate Bacillus anthracis strains.

    Science.gov (United States)

    Brumlik, M J; Szymajda, U; Zakowska, D; Liang, X; Redkar, R J; Patra, G; Del Vecchio, V G

    2001-07-01

    The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105 B. anthracis strains of diverse geographical origins. All B. anthracis strains produced fingerprints comprising seven to eight bands, referred to as "skeleton" bands, while one to three "diagnostic" bands differentiated between B. anthracis strains. LR REP-PCR fingerprints of B. anthracis strains showed very little in common with those of other closely related species such as B. cereus, B. thuringiensis, and B. mycoides, suggesting relative heterogeneity among the non-B. anthracis strains. Fingerprints from transitional non-B. anthracis strains, which possessed the B. anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B. anthracis groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating B. anthracis strains.

  16. Bacillus cereus G9241 Makes Anthrax Toxin and Capsule like Highly Virulent B. anthracis Ames but Behaves like Attenuated Toxigenic Nonencapsulated B. anthracis Sterne in Rabbits and Mice

    Science.gov (United States)

    2011-08-01

    Microbiology. All Rights Reserved. Bacillus cereus G9241 Makes Anthrax Toxin and Capsule like Highly Virulent B. anthracis Ames but Behaves like...G9241 for mice requires the presence of both plasmids. The Bacillus cereus group, of which Bacillus anthracis, Bacil- lus thuringiensis , and B... Bacillus cereus G9241 Makes Anthrax Toxin and Capsule like Highly Virulent B. anthracis Ames but Behaves like Attenuated Toxigenic Nonencapsulated B

  17. Novel and unique diagnostic biomarkers for Bacillus anthracis infection.

    Science.gov (United States)

    Sela-Abramovich, Sagit; Chitlaru, Theodor; Gat, Orit; Grosfeld, Haim; Cohen, Ofer; Shafferman, Avigdor

    2009-10-01

    A search for bacterium-specific biomarkers in peripheral blood following infection with Bacillus anthracis was carried out with rabbits, using a battery of specific antibodies generated by DNA vaccination against 10 preselected highly immunogenic bacterial antigens which were identified previously by a genomic/proteomic/serologic screen of the B. anthracis secretome. Detection of infection biomarkers in the circulation of infected rabbits could be achieved only after removal of highly abundant serum proteins by chromatography using a random-ligand affinity column. Besides the toxin component protective antigen, the following three secreted proteins were detected in the circulation of infected animals: the chaperone and protease HtrA (BA3660), an NlpC/P60 endopeptidase (BA1952), and a protein of unknown function harboring two SH3 (Src homology 3) domains (BA0796). The three proteins could be detected in plasma samples from infected animals exhibiting 10(3) to 10(5) CFU/ml blood and also in standard blood cultures at 3 to 6 h post-bacterial inoculation at a bacteremic level as low as 10(3) CFU/ml. Furthermore, the three biomarkers appear to be present only in the secretome of B. anthracis, not in those of the related pathogens B. thuringiensis and B. cereus. To the best of our knowledge, this is the first report of direct detection of B. anthracis-specific proteins, other than the toxin components, in the circulation of infected animals.

  18. A selective chromogenic agar that distinguishes Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis.

    Science.gov (United States)

    Juergensmeyer, Margaret A; Gingras, Bruce A; Restaino, Lawrence; Frampton, Elon W

    2006-08-01

    A selective and differential plating medium, R & F anthracis chromogenic agar (ACA), has been developed for isolating and identifying presumptive colonies of Bacillus anthracis. ACA contains the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-choline phosphate that upon hydrolysis yields teal (blue green) colonies indicating the presence of phosphatidylcholine-specific phospholipase C (PC-PLC) activity. Among seven Bacillus species tested on ACA, only members of the Bacillus cereus group (B. anthracis, B. cereus, and B. thuringiensis) produced teal colonies (PC-PLC positive) having cream rings. Examination of colony morphology in 18 pure culture strains of B. anthracis (15 ATCC strains plus AMES-1-RIID, ANR-1, and AMED-RIID), with one exception, required 48 h at 35 to 37 degrees C for significant color production, whereas only 24 h was required for B. cereus and B. thuringiensis. This differential rate of PC-PLC synthesis in B. anthracis (due to the truncated plcR gene and PlcR regulator in B. anthracis) allowed for the rapid differentiation on ACA of presumptive colonies of B. anthracis from B. cereus and B. thuringiensis in both pure and mixed cultures. Effective recovery of B. anthracis from a variety of matrices having both high (soil and sewage) and low microbial backgrounds (cloth, paper, and blood) spiked with B. anthracis ANR-1 spores suggests the probable utility of ACA plating for B. anthracis recovery in a diversity of applications.

  19. The function of PlcR in Bacillus anthracis vaccine strain A16R.

    Science.gov (United States)

    Xiaolin, Jia; Dongshu, Wang; Zhiqi, Gao; Erling, Feng; Jiping, Zheng; Hengliang, Wang; Guiying, Guo; Xiankai, Liu

    2015-05-01

    Bacillus anthracis, B. thuringiensis and B. cereus are members of the B. cereus group. They share high genetic similarity. Whereas plcR (Phospholipase C regulator) usually encodes a functional pleiotropic activator protein in B. cereus and B. thuringiensis isolates, a characteristic nonsense mutation is found in all B. anthracis strains investigated, making the gene dysfunctional. To study the function of PlcR in B. anthracis, we used the B. cereus CMCC63301 genome as a template and constructed a recombinant expression plasmid pBE2A-plcR, and introduced it into the B. anthracis vaccine strain A16R, and then analyzed the activity of the hemolysin and sphingomyelinase. The results showed that transformation of B. anthracis with plasmid pBE2A-plcR carrying the native B. cereus plcR gene active the expression of sphingomyelinase gene, but did not activate expression of hemolysin genes of B. anthracis A16R.

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

    Science.gov (United States)

    2012-06-13

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

  1. Global gene expression by Bacillus anthracis during growth in mammalian blood.

    Science.gov (United States)

    Carlson, Paul E; Bourgis, Alexandra E T; Hagan, Ada K; Hanna, Philip C

    2015-11-01

    During the late stages of systemic anthrax, Bacillus anthracis grows rapidly in the host bloodstream. To identify potential genes necessary for this observed rapid growth, we defined the transcriptional profile of B. anthracis during in vitro growth in bovine blood. Genome-wide transcriptome analysis indicated that B. anthracis undergoes significant changes in its transcriptome profile during growth in blood, including the differential regulation of genes associated both with metabolism and known virulence factors. Collectively, these data provide a framework for future studies identifying specific B. anthracis factors required for growth in the mammalian bloodstream.

  2. Characterization of Genetic Diversity of Bacillus anthracis in France by Using High-Resolution Melting Assays and Multilocus Variable-Number Tandem-Repeat Analysis ▿ †

    Science.gov (United States)

    Derzelle, S.; Laroche, S.; Le Flèche, P.; Hauck, Y.; Thierry, S.; Vergnaud, G.; Madani, N.

    2011-01-01

    Using high-resolution melting (HRM) analysis, we developed a cost-effective method to genotype a set of 13 phylogenetically informative single-nucleotide polymorphisms (SNPs) within the genome of Bacillus anthracis. SNP discrimination assays were performed in monoplex or duplex and applied to 100 B. anthracis isolates collected in France from 1953 to 2009 and a few reference strains. HRM provided a reliable and cheap alternative to subtype B. anthracis into one of the 12 major sublineages or subgroups. All strains could be correctly positioned on the canonical SNP (canSNP) phylogenetic tree, except the divergent Pasteur vaccine strain ATCC 4229. We detected the cooccurrence of three canSNP subgroups in France. The dominant B.Br.CNEVA sublineage was found to be prevalent in the Alps, the Pyrenees, the Auvergne region, and the Saône-et-Loire department. Strains affiliated with the A.Br.008/009 subgroup were observed throughout most of the country. The minor A.Br.001/002 subgroup was restricted to northeastern France. Multiple-locus variable-number tandem-repeat analysis using 24 markers further resolved French strains into 60 unique profiles and identified some regional patterns. Diversity found within the A.Br.008/009 and B.Br.CNEVA subgroups suggests that these represent old, ecologically established clades in France. Phylogenetic relationships with strains from other parts of the world are discussed. PMID:21998431

  3. 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...

  4. Photothermal spectroscopy of Bacillus anthracis and Bacillus cereus with microcantilevers

    Energy Technology Data Exchange (ETDEWEB)

    Wig, Andrew G [ORNL; Arakawa, Edward T [ORNL; Passian, Ali [ORNL; Ferrell, Thomas L [ORNL; Thundat, Thomas George [ORNL

    2006-03-01

    Microcalorimetric optical and infrared spectroscopy is a method of determining the spectral absorption of small quantities of materials over a wide range of incident wavelengths. In this paper, the first spectroscopic results for microcantilevers coated with Bacillus anthracis (BA) are presented. These results, for B. anthracis from 2.5 to 14.5 {micro}m, are compared with results from microcantilevers coated with Bacillus cereus (BC) and standard spectroscopic absorption data. The results demonstrate strong correlation between the deflection measurements and the reference spectroscopic absorption peaks. An advantage of this microcantilever-based method over traditional spectroscopy is that much smaller amounts of material (nanogram quantities) can be detected in comparison with the milligram amounts needed for standard methods. Another advantage is that the complete system can be relatively small without sacrificing spectral resolution.

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

    OpenAIRE

    2016-01-01

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

  6. Microarray-based Resequencing of Multiple Bacillus anthracis Isolates

    Science.gov (United States)

    2004-12-17

    al.: Iden- tification of anthrax toxin genes in a Bacillus cereus associ- ated with an illness resembling inhalation anthrax. Proc Natl Acad Sci USA...Norwegian Bacillus cereus and Bacillus thuringiensis soil isolates. Appl Environ Microbiol 2001, 67:4863-4873. 26. Radnedge L, Agron PG, Hill KK, Jackson PJ...Ticknor LO, Keim P, Andersen GL: Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis . Appl

  7. Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh.

    Science.gov (United States)

    Rume, Farzana Islam; Affuso, Alessia; Serrecchia, Luigina; Rondinone, Valeria; Manzulli, Viviana; Campese, Emanuele; Di Taranto, Pietro; Biswas, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Yasmin, Mahmuda; Fasanella, Antonio; Hugh-Jones, Martin

    2016-01-01

    In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.

  8. Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis

    Science.gov (United States)

    2011-09-01

    Army Research Laboratory: 2010; p 19. 18. Sambrook, J.; Fritsch, E. F.; Maniatis , T. Molecular Cloning : A Laboratory Manual. 2nd ed.; Cold Spring...on a rotating platform until not viscous (adapted from Molecular Cloning : A Laboratory Manual) (18). Each solution was centrifuged at 45,000 x g for... Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis by Deborah A. Sarkes, Joshua M. Kogot, Irene Val-Addo

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

    Science.gov (United States)

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

    2010-10-01

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

  10. 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.

  11. Molecular characterization of the circulating Bacillus anthracis in Jordan.

    Science.gov (United States)

    Aqel, Amin Abdelfattah; Hailat, Ekhlas; Serrecchia, Luigina; Aqel, Suad; Campese, Emanuele; Vicari, Nadia; Fasanella, Antonio

    2015-12-01

    To understand the biomolecular charcteristics of Bacillus anthracis in Jordan, 20 blood smear slides from dead animals with suspected anthrax were analyzed using conventional and molecular approaches. All slides were positive for B. anthracis by conventional staining but no growth of the organism on selective media was detected. However, of the 20 samples, 16 were B. anthracis DNA-positive using polymerase chain reaction (PCR). Seven samples provided enough quantity and quality of DNA, and their multilocus variable tandem repeat analysis (MLVA)-15 loci analysis revealed two different genotypes. All genotypes were belonging to A.B..r. 008/009 which is very common in Asia and Europe. Single nucleotide repeat (SNR) analysis revealed that there were no sub genotypes. Molecular diagnosis of animal anthrax in Jordan is not used routinely; henceforth, official diagnosis of anthrax is based on the observation of the slides by optical microscope and this can often cause reading errors. Therefore, the prevalence of the disease in Jordan might be slightly lower than that reported by the official bodies.

  12. The Bacillus anthracis Exosporium: What's the Big "Hairy" Deal?

    Science.gov (United States)

    Bozue, Joel A; Welkos, Susan; Cote, Christopher K

    2015-10-01

    In some Bacillus species, including Bacillus subtilis, the coat is the outermost layer of the spore. In others, such as the Bacillus cereus family, there is an additional layer that envelops the coat, called the exosporium. In the case of Bacillus anthracis, a series of fine hair-like projections, also referred to as a "hairy" nap, extends from the exosporium basal layer. The exact role of the exosporium in B. anthracis, or for any of the Bacillus species possessing this structure, remains unclear. However, it has been assumed that the exosporium would play some role in infection for B. anthracis, because it is the outermost structure of the spore and would make initial contact with host and immune cells during infection. Therefore, the exosporium has been a topic of great interest, and over the past decade much progress has been made to understand its composition, biosynthesis, and potential roles. Several key aspects of this spore structure, however, are still debated and remain undetermined. Although insights have been gained on the interaction of exosporium with the host during infection, the exact role and significance of this complex structure remain to be determined. Furthermore, because the exosporium is a highly antigenic structure, future strategies for the next-generation anthrax vaccine should pursue its inclusion as a component to provide protection against the spore itself during the initial stages of anthrax.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

  15. Phylogeography of Bacillus anthracis in the country of Georgia shows evidence of population structuring and is dissimilar to other regional genotypes.

    Directory of Open Access Journals (Sweden)

    Ekaterine Khmaladze

    Full Text Available Sequence analyses and subtyping of Bacillus anthracis strains from Georgia reveal a single distinct lineage (Aust94 that is ecologically established. Phylogeographic analysis and comparisons to a global collection reveals a clade that is mostly restricted to Georgia. Within this clade, many groups are found around the country, however at least one subclade is only found in the eastern part. This pattern suggests that dispersal into and out of Georgia has been rare and despite historical dispersion within the country, for at least for one lineage, current spread is limited.

  16. Genome Sequence of the Soviet/Russian Bacillus anthracis Vaccine Strain 55-VNIIVViM

    Science.gov (United States)

    Kotorashvili, Adam

    2016-01-01

    Bacillus anthracis strain 55-VNIIVViM is a live-attenuated nonencapsulated Soviet/Russian veterinary anthrax vaccine strain. We report here the genome of 55-VNIIVViM and confirm its phylogenetic placement in the global population structure of B. anthracis. PMID:28007853

  17. The Pathogenomic Sequence Analysis of B. cereus and B. Thuringiensis isolates closely related to Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Han, C S; Xie, G; Challacombe, J F; Altherr, M R; Bhotika, S S; Bruce, D; Campbell, C S; Campbell, M L; Chen, J; Chertkov, O; Cleland, C; Dimitrijevic-Bussod, M; Doggett, N A; Fawcett, J J; Glavina, T; Goodwin, L A; Hill, K K; Hitchcock, P; Jackson, P J; Keim, P; Kewalramani, A R; Longmire, J; Lucas, S; Malfatti, S; McMurry, K; Meincke, L J; Misra, M; Moseman, B L; Mundt, M; Munk, A C; Okinaka, R T; Parson-Quintana, B; Reilly, L P; Richardson, P; Robinson, D L; Rubin, E; Saunders, E; Tapia, R; Tesmer, J G; Thayer, N; Thompson, L S; Tice, H; Ticknor, L O; Wills, P L; Gilna, P; Brettin, T S

    2005-10-12

    The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B. cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including B anthracis. Comparative analysis of these two genomes with other members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.

  18. Pathogenic ecology: Where have all the pathogens gone? Anthrax: a classic case

    Science.gov (United States)

    Kiel, Johnathan; Walker, Wes W.; Andrews, Carrie J.; De Los Santos, Amy; Adams, Roy N.; Bucholz, Matthew W.; McBurnett, Shelly D.; Fuentes, Vladimir; Rizner, Karon E.; Blount, Keith W.

    2009-05-01

    Pathogenic ecology is the natural relationship to animate and inanimate components of the environment that support the sustainment of a pathogen in the environment or prohibit its sustainment, or their interactions with an introduced pathogen that allow for the establishment of disease in a new environment. The anthrax bacterium in the spore form has been recognized as a highly likely biological warfare or terrorist agent. The purpose of this work was to determine the environmental reservoir of Bacillus anthracis between outbreaks of anthrax and to examine the potential factors influencing the conversion of the Bacillus anthracis from a quiescent state to the disease causing state. Here we provide environmental and laboratory data for the cycling of Bacillus anthracis in plants to reconcile observations that contradict the soil borne hypothesis of anthrax maintenance in the environment.

  19. A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species.

    Directory of Open Access Journals (Sweden)

    Hirohito Ogawa

    Full Text Available Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.

  20. A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species.

    Science.gov (United States)

    Ogawa, Hirohito; Fujikura, Daisuke; Ohnuma, Miyuki; Ohnishi, Naomi; Hang'ombe, Bernard M; Mimuro, Hitomi; Ezaki, Takayuki; Mweene, Aaron S; Higashi, Hideaki

    2015-01-01

    Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.

  1. Nitric Oxide as a Regulator of B. anthracis Pathogenicity

    Directory of Open Access Journals (Sweden)

    Serguei G Popov

    2015-09-01

    Full Text Available Nitric oxide (NO is a key physiological regulator in eukaryotic and prokaryotic organisms. It can cause a variety of biological effects by reacting with its targets or/and indirectly inducing oxidative stress. NO can also be produced by bacteria including the pathogenic B. anthracis; however its role in the infectious process only begins to emerge. NO incapacitates macrophages by S-nitrosylating the intracellular proteins and protects B. anthracis from oxidative stress. It is also implicated in the formation of toxic peroxynitrite. In this study we further assessed the effects of B. anthracis NO produced by the NO synthase (bNOS on bacterial metabolism and host cells in experiments with the bNOS knockout Sterne strain. The mutation abrogated accumulation of nitrite and nitrate as tracer products of NO in the culture medium and markedly attenuated growth in both aerobic and microaerobic conditions. The regulatory role of NO was also suggested by the abnormally high rate of nitrate denitrification by the mutant in the presence of oxygen. Anaerobic regulation mediated by NO was reflected in reduced fermentation of glucose by the mutant correlating with the reduced toxicity of bacteria toward host cells in culture. The toxic effect of NO required permeabilization of the target cells as well as the activity of fermentation-derived metabolite in the conditions of reduced pH. The host cells demonstrated increased phosphorylation of major survivor protein kinase AKT correlating with reduced toxicity of the mutant in comparison with Sterne. Our global proteomic analysis of lymph from the lymph nodes of infected mice harboring bacteria revealed numerous changes in the pattern and levels of proteins associated with the activity of bNOS influencing key cell physiological processes relevant to energy metabolism, growth, signal transduction, stress response, septic shock and homeostasis. This is the first in vivo observation of the bacterial NO effect on the

  2. Antimicrobial susceptibility of Bacillus anthracis strains from Hungary.

    Science.gov (United States)

    Kreizinger, Zsuzsa; Sulyok, Kinga Mária; Makrai, László; Rónai, Zsuzsanna; Fodor, László; Jánosi, Szilárd; Gyuranecz, Miklós

    2016-06-01

    The susceptibility of 29 Bacillus anthracis strains, collected in Hungary between 1933 and 2014, was tested to 10 antibiotics with commercially available minimum inhibitory concentration (MIC) test strips. All strains were susceptible to amoxicillin, ciprofloxacin, clindamycin, doxycycline, gentamicin, penicillin, rifampicin, and vancomycin. Intermediate susceptibility to erythromycin and cefotaxime was detected in 17.2% (5/29) and 58.6% (17/29) of the strains, respectively. Correlations were not observed between the isolation date, location, host species, genotype, and antibiotic susceptibility profile of strains.

  3. Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization

    Directory of Open Access Journals (Sweden)

    Redkar Rajendra

    2004-02-01

    Full Text Available Abstract Background Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH was used to identify specific chromosomal sequences unique to B. anthracis. Results Two SSH libraries were generated. Genomic DNA from plasmid-cured B. anthracis was used as the tester DNA in both libraries, while genomic DNA from either B. cereus or B. thuringiensis served as the driver DNA. Progressive screening of the libraries by colony filter and Southern blot analyses identified 29 different clones that were specific for the B. anthracis chromosome relative not only to the respective driver DNAs, but also to seven other different strains of B. cereus and B. thuringiensis included in the process. The nucleotide sequences of the clones were compared with those found in genomic databases, revealing that over half of the clones were located into 2 regions on the B. anthracis chromosome. Conclusions Genes encoding potential cell wall synthesis proteins dominated one region, while bacteriophage-related sequences dominated the other region. The latter supports the hypothesis that acquisition of these bacteriophage sequences occurred during or after speciation of B. anthracis relative to B. cereus and B. thuringiensis. This study provides insight into the chromosomal differences between B. anthracis and its closest phylogenetic relatives.

  4. [Ecology and ecologies].

    Science.gov (United States)

    Valera, Luca

    2011-01-01

    Ecology (from the Greek words οιχοσ, "house" and λογια "study of") is the science of the "house", since it studies the environments where we live. There are three main ways of thinking about Ecology: Ecology as the study of interactions (between humans and the environment, between humans and living beings, between all living beings, etc.), Ecology as the statistical study of interactions, Ecology as a faith, or rather as a science that requires a metaphysical view. The history of Ecology shows us how this view was released by the label of "folk sense" to gain the epistemological status of science, a science that strives to be interdisciplinary. So, the aim of Ecology is to study, through a scientific methodology, the whole natural world, answering to very different questions, that arise from several fields (Economics, Biology, Sociology, Philosophy, etc.). The plurality of issues that Ecology has to face led, during the Twentieth-century, to branch off in several different "ecologies". As a result, each one of these new approaches chose as its own field a more limited and specific portion of reality.

  5. Characterization of the sortase repertoire in Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Willy Aucher

    Full Text Available LPXTG proteins, present in most if not all Gram-positive bacteria, are known to be anchored by sortases to the bacterial peptidoglycan. More than one sortase gene is often encoded in a bacterial species, and each sortase is supposed to specifically anchor given LPXTG proteins, depending of the sequence of the C-terminal cell wall sorting signal (cwss, bearing an LPXTG motif or another recognition sequence. B. anthracis possesses three sortase genes. B. anthracis sortase deleted mutant strains are not affected in their virulence. To determine the sortase repertoires, we developed a genetic screen using the property of the gamma phage to lyse bacteria only when its receptor, GamR, an LPXTG protein, is exposed at the surface. We identified 10 proteins that contain a cell wall sorting signal and are covalently anchored to the peptidoglycan. Some chimeric proteins yielded phage lysis in all sortase mutant strains, suggesting that cwss proteins remained surface accessible in absence of their anchoring sortase, probably as a consequence of membrane localization of yet uncleaved precursor proteins. For definite assignment of the sortase repertoires, we consequently relied on a complementary test, using a biochemical approach, namely immunoblot experiments. The sortase anchoring nine of these proteins has thus been determined. The absence of virulence defect of the sortase mutants could be a consequence of the membrane localization of the cwss proteins.

  6. Development of internal controls for PCR detection of Bacillus anthracis.

    Science.gov (United States)

    Brightwell, G; Pearce, M; Leslie, D

    1998-12-01

    This work describes the development and evaluation of a multiplex polymerase chain reaction (PCR) for the detection of Bacillus anthracis strains harbouring plasmid pX02. The multiplex also incorporated an internal control (IC) to avoid false negative reactions. Internal controls consisted of plasmids containing modified PCR target sequences, corresponding to the capC and BA813 genes of B. anthracis, which were then co-amplified with the original target sequences using the same set of amplimers. The initial IC construct comprised of an internally deleted form of the genomic target sequence cloned into pUC19. A series of nested DNA fragments corresponding to the 23S rRNA sequences of Bacillus cereus were then subcloned into the point of deletion, producing a number of IC constructs with similar sequences but increasing product size on PCR amplification. Neither the presence of IC DNA template or IC PCR product size affected the specificity or non-specific cross-reactivity of the original PCR assay. The concentration of IC was critical, too much IC DNA template would out compete the genomic DNA template, thus giving a false negative result. However, when the concentration of IC was optimal assay sensitivity was not compromised.

  7. [Bacillus anthracis: a molecular look at a famous pathogen].

    Science.gov (United States)

    Pavan, María E; Pettinari, María J; Cairó, Fabián; Pavan, Esteban E; Cataldi, Angel A

    2011-01-01

    Bacillus anthracis, a gram-positive rod belonging to the Bacillus cereus group, has an extremely monomorphic genome, and presents high structural and physiological similarity with B. cereus and Bacillus thuringiensis. In this work, the new molecular methods for the identification and typing of B. anthracis developed in the last years, based on variable number tandem repeats or on genetic differences detected through sequencing, are described. The molecular aspects of traditional virulence factors: capsule, protective antigen, lethal factor and edema factor are described in depth, together with virulence factors recently proposed, such as the siderophores petrobactin and bacillibactin, the S-layer adhesin and the MntA lipoprotein. It is detailed the molecular organization of megaplasmids pXO1 and pXO2, including the pathogenicity island of pXO1. The genetic skeleton of these plasmids has been observed in related species, and this could be attributed to lateral gene transfer. Finally, the two anthrax toxin protective antigen receptors, ANTXR1/TEM8 and ANTXR2/CMG2, essential for the interaction of the pathogen with the host, are presented. The molecular studies performed in recent years have greatly increased knowledge in different aspects of this microorganism and its relationship with the host, but at the same time they have raised new questions about this noted pathogen.

  8. Bacillus anthracis genome organization in light of whole transcriptome sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Jeffrey; Zhu, Wenhan; Passalacqua, Karla D.; Bergman, Nicholas; Borodovsky, Mark

    2010-03-22

    Emerging knowledge of whole prokaryotic transcriptomes could validate a number of theoretical concepts introduced in the early days of genomics. What are the rules connecting gene expression levels with sequence determinants such as quantitative scores of promoters and terminators? Are translation efficiency measures, e.g. codon adaptation index and RBS score related to gene expression? We used the whole transcriptome shotgun sequencing of a bacterial pathogen Bacillus anthracis to assess correlation of gene expression level with promoter, terminator and RBS scores, codon adaptation index, as well as with a new measure of gene translational efficiency, average translation speed. We compared computational predictions of operon topologies with the transcript borders inferred from RNA-Seq reads. Transcriptome mapping may also improve existing gene annotation. Upon assessment of accuracy of current annotation of protein-coding genes in the B. anthracis genome we have shown that the transcriptome data indicate existence of more than a hundred genes missing in the annotation though predicted by an ab initio gene finder. Interestingly, we observed that many pseudogenes possess not only a sequence with detectable coding potential but also promoters that maintain transcriptional activity.

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

    Science.gov (United States)

    Nicholson, Wayne L; Galeano, Belinda

    2003-02-01

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

  10. Selective detection of 1000 B. anthracis spores within 15 minutes using a peptide functionalized SERS assay.

    Science.gov (United States)

    Farquharson, Stuart; Shende, Chetan; Smith, Wayne; Huang, Hermes; Inscore, Frank; Sengupta, Atanu; Sperry, Jay; Sickler, Todd; Prugh, Amber; Guicheteau, Jason

    2014-12-21

    A surface-enhanced Raman spectroscopy (SERS) assay has been designed to detect Bacillus anthracis spores. The assay consists of silver nanoparticles embedded in a porous glass structure that have been functionalized with ATYPLPIR, a peptide developed to discriminately bind B. anthracis versus other species of Bacillus. Once bound, acetic acid was used to release the biomarker dipicolinic acid from the spores, which was detected by SERS through the addition of silver colloids. This SERS assay was used to selectively bind B. anthracis with a 100-fold selectivity versus B. cereus, and to detect B. anthracis Ames at concentrations of 1000 spores per mL within 15 minutes. The SERS assay measurements provide a basis for the development of systems that can detect spores collected from the air or from water supplies.

  11. Colonic immune suppression, barrier dysfunction, and dysbiosis by gastrointestinal bacillus anthracis Infection.

    Directory of Open Access Journals (Sweden)

    Yaíma L Lightfoot

    Full Text Available Gastrointestinal (GI anthrax results from the ingestion of Bacillus anthracis. Herein, we investigated the pathogenesis of GI anthrax in animals orally infected with toxigenic non-encapsulated B. anthracis Sterne strain (pXO1+ pXO2- spores that resulted in rapid animal death. B. anthracis Sterne induced significant breakdown of intestinal barrier function and led to gut dysbiosis, resulting in systemic dissemination of not only B. anthracis, but also of commensals. Disease progression significantly correlated with the deterioration of innate and T cell functions. Our studies provide critical immunologic and physiologic insights into the pathogenesis of GI anthrax infection, whereupon cleavage of mitogen-activated protein kinases (MAPKs in immune cells may play a central role in promoting dysfunctional immune responses against this deadly pathogen.

  12. 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...

  13. WalRK two component system of Bacillus anthracis responds to temperature and antibiotic stress.

    Science.gov (United States)

    Dhiman, Alisha; Gopalani, Monisha; Bhatnagar, Rakesh

    2015-04-17

    WalRK Two Component System (TCS) of Bacillus anthracis forms a functional TCS. This report elaborates upon the WalRK genomic architecture, promoter structure, promoter activity and expression under various stress conditions in B. anthracis. 5' RACE located the WalRK functional promoter within 317 bp region upstream of WalR. Reporter gene assays demonstrated maximal promoter activity during early growth phases indicating utility in exponential stages of growth. qRT-PCR showed upregulation of WalRK transcripts during temperature and antibiotic stress. However, WalR overexpression did not affect the tested antibiotic MIC values in B. anthracis. Collectively, these results confirm that WalRK responds to cell envelope stress in B. anthracis.

  14. Impedance Measurements Could Accelerate Phage-Based Identification of Bacillus anthracis and Other Bacteria

    Science.gov (United States)

    2016-09-01

    Impedance Measurements Could Accelerate Phage-Based Identification of Bacillus anthracis And Other Bacteria Thomas Brown, Salwa Shan, Teresa...This is particularly true in the field of biodefense where phage have a long history of being used to identify Bacillus anthracis and Yersinia pestis...based diagnostic assays for this pathogen. After exposing small quantities of Bacillus cultures to ɣ phage, we tracked the cultures for up to 90

  15. Crossing of the epithelial barriers by Bacillus anthracis: the Known and the Unknown

    OpenAIRE

    2015-01-01

    Anthrax, caused by Bacillus anthracis, a Gram-positive spore-forming bacterium, is initiated by the entry of spores into the host body. There are three types of human infection: cutaneous, inhalational, and gastrointestinal. For each form, B. anthracis spores need to cross the cutaneous, respiratory or digestive epithelial barriers, respectively, as a first obligate step to establish infection. Anthrax is a toxi-infection: an association of toxemia and rapidly spreading infection progressing ...

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

    Science.gov (United States)

    2006-01-01

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

  17. Characterization of Anthrolysin O, the Bacillus anthracis Cholesterol-Dependent Cytolysin

    OpenAIRE

    Shannon, Jeffrey G; Ross, Cana L.; Koehler, Theresa M.; Rest, Richard F

    2003-01-01

    We characterized the expression of a putative toxin of Bacillus anthracis, a member of the cholesterol-dependent cytolysin (CDC) family, which includes listeriolysin O, perfringolysin O, and streptolysin O. We named this cytotoxin anthrolysin O (ALO). Although B. anthracis expresses minimal hemolytic activity in clinical settings, we show that Sterne strain 7702 expresses hemolytic activity when grown in brain heart infusion broth or in other rich bacteriologic media, but it secretes barely d...

  18. Indirect Detection Of Bacillus Anthracis (Anthrax) Using Amplified Gamma Phage-Based Assays

    Science.gov (United States)

    2007-11-01

    Figure 3.3 Pasteur Institute TEM of Bacillus surface 31 Bacillus anthracis is taxonomically aligned with B. cereus , B. thuringiensis and B...None of the DNA from bacteria (B. anthracis, B. cereus , Staphylococcus aureus, Pseudomonas aeroginosa, and Neisseria gonorrhea), yeast, blood , or...49-54. 59. Ryzhov, V., Y. Hathout, and C. Fenselau, Rapid Characterization of Spores of Bacillus Cereus Group Bacteria by Matrix-assisted Laser

  19. Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro

    Science.gov (United States)

    2013-03-11

    Bacillus cereus group of bacteria, are attributed to poly- γ-D-glutamate acid (PGA) capsule, lethal toxin (LT) and edema toxin (ET) [10-12]. These toxins...M, Hellman M, Muhie S, et al. (2013) Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro...author and source are credited. Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro Rasha

  20. Use of Long-Range Repetitive Element Polymorphism-PCR To Differentiate Bacillus anthracis Strains

    OpenAIRE

    Brumlik, Michael J.; Szymajda, Urszula; Zakowska, Dorota; Liang, Xudong; Redkar, Rajendra J.; Patra, Guy; Del Vecchio, Vito G.

    2001-01-01

    The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 10...

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

    Science.gov (United States)

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

    2014-03-01

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

  2. The Bacillus anthracis chromosome contains four conserved, excision-proficient, putative prophages

    Directory of Open Access Journals (Sweden)

    Sozhamannan Shanmuga

    2006-04-01

    Full Text Available Abstract Background Bacillus anthracis is considered to be a recently emerged clone within the Bacillus cereus sensu lato group. The B. anthracis genome sequence contains four putative lambdoid prophages. We undertook this study in order to understand whether the four prophages are unique to B. anthracis and whether they produce active phages. Results More than 300 geographically and temporally divergent isolates of B. anthracis and its near neighbors were screened by PCR for the presence of specific DNA sequences from each prophage region. Every isolate of B. anthracis screened by PCR was found to produce all four phage-specific amplicons whereas none of the non-B. anthracis isolates, produced more than one phage-specific amplicon. Excision of prophages could be detected by a PCR based assay for attP sites on extra-chromosomal phage circles and for attB sites on phage-excised chromosomes. SYBR-green real-time PCR assays indicated that prophage excision occurs at very low frequencies (2 × 10-5 - 8 × 10-8/cell. Induction with mitomycin C increased the frequency of excision of one of the prophages by approximately 250 fold. All four prophages appear to be defective since, mitomycin C induced culture did not release any viable phage particle or lyse the cells or reveal any phage particle under electron microscopic examination. Conclusion The retention of all four putative prophage regions across all tested strains of B. anthracis is further evidence of the very recent emergence of this lineage and the prophage regions may be useful for differentiating the B. anthracis chromosome from that of its neighbors. All four prophages can excise at low frequencies, but are apparently defective in phage production.

  3. Effects of Endogenous d-Alanine Synthesis and Autoinhibition of Bacillus anthracis Germination on In Vitro and In Vivo Infections▿

    OpenAIRE

    McKevitt, Matthew T.; Bryant, Katie M.; Shakir, Salika M.; Larabee, Jason L.; Blanke, Steven R.; Lovchik, Julie; Lyons, C. Rick; Ballard, Jimmy D.

    2007-01-01

    Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous d-alanine production to the efficiency and timing of B. anthracis spore germination under in vit...

  4. 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.

  5. Capsules, toxins and AtxA as virulence factors of emerging Bacillus cereus biovar anthracis.

    Science.gov (United States)

    Brézillon, Christophe; Haustant, Michel; Dupke, Susann; Corre, Jean-Philippe; Lander, Angelika; Franz, Tatjana; Monot, Marc; Couture-Tosi, Evelyne; Jouvion, Gregory; Leendertz, Fabian H; Grunow, Roland; Mock, Michèle E; Klee, Silke R; Goossens, Pierre L

    2015-04-01

    Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain) and Côte d'Ivoire (CI strain). These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA) capsule and the B. anthracis polyglutamate (PDGA) capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have emerged.

  6. Administrative Ecology

    Science.gov (United States)

    McGarity, Augustus C., III; Maulding, Wanda

    2007-01-01

    This article discusses how all four facets of administrative ecology help dispel the claims about the "impossibility" of the superintendency. These are personal ecology, professional ecology, organizational ecology, and community ecology. Using today's superintendency as an administrative platform, current literature describes a preponderance of…

  7. Comparison of French and Worldwide Bacillus anthracis Strains Favors a Recent, Post-Columbian Origin of the Predominant North-American Clade.

    Directory of Open Access Journals (Sweden)

    Gilles Vergnaud

    Full Text Available Bacillus anthracis, the highly dangerous zoonotic bacterial pathogen species is currently composed of three genetic groups, called A, B and C. Group A is represented worldwide whereas group B is present essentially in Western Europe and Southern Africa. Only three strains from group C have been reported. This knowledge is derived from the genotyping of more than 2000 strains collected worldwide. Strains from both group A and group B are present in France. Previous investigations showed that the majority of sporadic French strains belong to the so-called A.Br.011/009 group A clade and define a very remarkable polytomy with six branches. Here we explore the significance of this polytomy by comparing the French B. anthracis lineages to worldwide lineages. We take advantage of whole genome sequence data previously determined for 122 French strains and 45 strains of various origins.A total of 6690 SNPs was identified among the available dataset and used to draw the phylogeny. The phylogeny of the French B group strains which belongs to B.Br.CNEVA indicates an expansion from the south-east part of France (the Alps towards the south-west (Massif-Central and Pyrenees. The relatively small group A strains belonging to A.Br.001/002 results from at least two independent introductions. Strikingly, the data clearly demonstrates that the currently predominant B. anthracis lineage in North America, called WNA for Western North American, is derived from one branch of the A.Br.011/009 polytomy predominant in France.The present work extends the range of observed substitution rate heterogeneity within B. anthracis, in agreement with its ecology and in contrast with some other pathogens. The population structure of the six branches A.Br.011/009 polytomy identified in France, diversity of branch length, and comparison with the WNA lineage, suggests that WNA is of post-Columbian and west European origin, with France as a likely source. Furthermore, it is tempting to

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

    OpenAIRE

    2012-01-01

    Bacillus anthracis, the bacterium that causes anthrax, is an agent of bioterrorism. The most effective antimicrobial therapy for B. anthracis infections is unknown. An in vitro pharmacodynamic model of B. anthracis was used to compare the efficacies of simulated clinically prescribed regimens of moxifloxacin, linezolid, and meropenem with the “gold standards,” doxycycline and ciprofloxacin. Treatment outcomes for isogenic spore-forming and non-spore-forming strains of B. anthracis were compar...

  9. Detection of Bacillus anthracis DNA in complex soil and air samples using next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Nicholas A Be

    Full Text Available Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy.

  10. Protection of inactive intranasal ántrax vaccine to Bacillus anthracis infection

    Directory of Open Access Journals (Sweden)

    Adin Priadi

    2010-06-01

    Full Text Available Ánthrax is an endemic zoonotic disease distributed in many parts of Indonesia. Although vaccination program has been implemented in many areas, cases are still frequently reported. Farmers are reluctant to vaccinate their livestock since spore vaccine used in the field often cause side effects and death of the animals. To overcome this problem, an inactive vaccine composes of Bacillus anthracis toxins, cell wall and capsule subunits was developed. B. anthracis Sterne strain (34F2 was selected to produce toxins and cell walls. Local Bacillus anthracis isolated from Citaringgul was used to produce capsule as the Polymerase Chain Reaction (PCR revealed that this isolate poses cap gene encoding for capsule. Two vaccines compose of 15 μg toxoid, 30 μg of capsule, 15 μg of cell wall and 30 μg toxoid, 60 μg of capsule, 15 μg of cell walls were designated as vaccine I and vaccine II respectively. For each experiment, 10 mice were nasally immunized by placing 5 μl of vaccine into each nare 3 times at 2-week intervals. A group of 10 mice were unvaccinated and used as control. Blood was collected fortnightly to monitor antibody responses. All mice were challenged with 2 x 105 B. anthracis Sterne spores injected subcutaneously two weeks after the last vaccination. Two weeks after vaccination of antibodies to B. anthracis toxin, capsule and cell wall were detected in dot-blot assay. Mice that were immunised intranasally with chitosan adjuvanted vaccine developed high IgG responses in sera as detected by ELISA, and the response was dose dependent. Vaccine II gave better response than vaccine I. Vaccine I and II protected mice from challenge at a rate of 60 and 80% respectively. This results showed that intranasal B. anthracis vaccine composes of toxin, capsule and cell wall with chitosan as an adjuvant gave a good protection against B. anthracis Sterne spores challenge in mice.

  11. Application of in vivo induced antigen technology (IVIAT to Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Sean M Rollins

    Full Text Available In vivo induced antigen technology (IVIAT is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42; the bacteriophage holin gene BA4074; and pagA (pXO1-110. The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.

  12. Rapid Detection of Bacillus anthracis in Complex Food Matrices Using Phage-Mediated Bioluminescence.

    Science.gov (United States)

    Sharp, Natasha J; Vandamm, Joshua P; Molineux, Ian J; Schofield, David A

    2015-05-01

    Bacillus anthracis, the causative agent of anthrax, is considered a high-priority agent that may be used in a food-related terrorist attack because it can be contracted by ingestion and it also forms spores with heat and chemical resistance. Thus, novel surveillance methodologies to detect B. anthracis on adulterated foods are important for bioterrorism preparedness. We describe the development of a phage-based bioluminescence assay for the detection of B. anthracis on deliberately contaminated foods. We previously engineered the B. anthracis phage Wβ with genes encoding bacterial luciferase (luxA and luxB) to create a "light-tagged" reporter (Wβ::luxAB) that is able to rapidly detect B. anthracis by transducing a bioluminescent signal response. Here, we investigate the ability of Wβ::luxAB to detect B. anthracis Sterne, an attenuated select agent strain, in inoculated food (ground beef) and milk (2%, baby formula, and half and half) matrices after incubation with spores for 72 h at 4°C as per AOAC testing guidelines. The majority of B. anthracis bacilli remained in spore form, and thus were potentially infectious, within each of the liquid matrices for 14 days. Detection limits were 80 CFU/ml after 7 h of enrichment; sensitivity of detection increased to 8 CFU/ml when enrichment was extended to 16 h. The limit of detection in ground beef was 3.2 × 10(3) CFU/g after 7 h of enrichment, improving to 3.2 × 10(2) CFU/g after 16 h. Because the time to result is rapid and minimal processing is required, and because gastrointestinal anthrax can be fatal, the reporter technology displays promise for the protection of our food supply following a deliberate release of this priority pathogen.

  13. Bacillus anthracis-Like Bacteria and Other B. cereus Group Members in a Microbial Community Within the International Space Station : A Challenge for Rapid and Easy Molecular Detection of Virulent B. anthracis

    NARCIS (Netherlands)

    van Tongeren, Sandra P.; Roest, Hendrik I. J.; Degener, John E.; Harmsen, Hermie J. M.

    2014-01-01

    For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order f

  14. Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the international space station: a challenge for rapid and easy molecular detection of virulent B. anthracis.

    NARCIS (Netherlands)

    Tongeren, van S.P.; Roest, H.I.J.; Degener, J.E.; Harmsen, H.J.M.

    2014-01-01

    For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order f

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

    Science.gov (United States)

    2015-06-19

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

  16. Dendritic Cell Targeting of Bacillus anthracis Protective Antigen Expressed by Lactobacillus acidophilus Protects Mice from Lethal Challenge

    Science.gov (United States)

    2008-10-28

    Dendritic cell targeting of Bacillus anthracis protective antigen expressed by Lactobacillus acidophilus protects mice from lethal challenge M...lethal chal- lenge. A vaccine strategy was established by using Lactobacillus acidophilus to deliver Bacillus anthracis protective antigen (PA) via...include species of Lactobacillus , Lactococcus, Leuconostoc, Pedio- coccus, and Streptococcus. It is widely accepted that Lactobacillus species play a

  17. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    NARCIS (Netherlands)

    Agren, J.; Hamidjaja, R.A.; Hansen, T.; Ruuls, R.C.; Thierry, S.; Vigre, H.; Janse, I.; Sundström, A.; Segerman, B.; Koene, M.G.J.; Löfström, Ch.; Rotterdam, van B.; Derzelle, S.

    2013-01-01

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely o

  18. Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples.

    Science.gov (United States)

    Irenge, Léonid M; Durant, Jean-François; Tomaso, Herbert; Pilo, Paola; Olsen, Jaran S; Ramisse, Vincent; Mahillon, Jacques; Gala, Jean-Luc

    2010-11-01

    A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

  19. Bacillus anthracis in China and its relationship to worldwide lineages

    Directory of Open Access Journals (Sweden)

    Schupp James M

    2009-04-01

    Full Text Available Abstract Background The global pattern of distribution of 1033 B. anthracis isolates has previously been defined by a set of 12 conserved canonical single nucleotide polymorphisms (canSNP. These studies reinforced the presence of three major lineages and 12 sub-lineages and sub-groups of this anthrax-causing pathogen. Isolates that form the A lineage (unlike the B and C lineages have become widely dispersed throughout the world and form the basis for the geographical disposition of "modern" anthrax. An archival collection of 191 different B. anthracis isolates from China provides a glimpse into the possible role of Chinese trade and commerce in the spread of certain sub-lineages of this pathogen. Canonical single nucleotide polymorphism (canSNP and multiple locus VNTR analysis (MLVA typing has been used to examine this archival collection of isolates. Results The canSNP study indicates that there are 5 different sub-lineages/sub-groups in China out of 12 previously described world-wide canSNP genotypes. Three of these canSNP genotypes were only found in the western-most province of China, Xinjiang. These genotypes were A.Br.008/009, a sub-group that is spread across most of Europe and Asia; A.Br.Aust 94, a sub-lineage that is present in Europe and India, and A.Br.Vollum, a lineage that is also present in Europe. The remaining two canSNP genotypes are spread across the whole of China and belong to sub-group A.Br.001/002 and the A.Br.Ames sub-lineage, two closely related genotypes. MLVA typing adds resolution to the isolates in each canSNP genotype and diversity indices for the A.Br.008/009 and A.Br.001/002 sub-groups suggest that these represent older and established clades in China. Conclusion B. anthracis isolates were recovered from three canSNP sub-groups (A.Br.008/009, A.Br.Aust94, and A.Br.Vollum in the western most portion of the large Chinese province of Xinjiang. The city of Kashi in this province appears to have served as a crossroads

  20. Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.

  1. Bacillus anthracis interacts with plasmin(ogen to evade C3b-dependent innate immunity.

    Directory of Open Access Journals (Sweden)

    Myung-Chul Chung

    Full Text Available The causative agent of anthrax, Bacillus anthracis, is capable of circumventing the humoral and innate immune defense of the host and modulating the blood chemistry in circulation to initiate a productive infection. It has been shown that the pathogen employs a number of strategies against immune cells using secreted pathogenic factors such as toxins. However, interference of B. anthracis with the innate immune system through specific interaction of the spore surface with host proteins such as the complement system has heretofore attracted little attention. In order to assess the mechanisms by which B. anthracis evades the defense system, we employed a proteomic analysis to identify human serum proteins interacting with B. anthracis spores, and found that plasminogen (PLG is a major surface-bound protein. PLG efficiently bound to spores in a lysine- and exosporium-dependent manner. We identified α-enolase and elongation factor tu as PLG receptors. PLG-bound spores were capable of exhibiting anti-opsonic properties by cleaving C3b molecules in vitro and in rabbit bronchoalveolar lavage fluid, resulting in a decrease in macrophage phagocytosis. Our findings represent a step forward in understanding the mechanisms involved in the evasion of innate immunity by B. anthracis through recruitment of PLG resulting in the enhancement of anti-complement and anti-opsonization properties of the pathogen.

  2. Decontamination Efficacy and Skin Toxicity of Two Decontaminants against Bacillus anthracis.

    Science.gov (United States)

    Stratilo, Chad W; Crichton, Melissa K F; Sawyer, Thomas W

    2015-01-01

    Decontamination of bacterial endospores such as Bacillus anthracis has traditionally required the use of harsh or caustic chemicals. The aim of this study was to evaluate the efficacy of a chlorine dioxide decontaminant in killing Bacillus anthracis spores in solution and on a human skin simulant (porcine cadaver skin), compared to that of commonly used sodium hypochlorite or soapy water decontamination procedures. In addition, the relative toxicities of these decontaminants were compared in human skin keratinocyte primary cultures. The chlorine dioxide decontaminant was similarly effective to sodium hypochlorite in reducing spore numbers of Bacillus anthracis Ames in liquid suspension after a 10 minute exposure. After five minutes, the chlorine dioxide product was significantly more efficacious. Decontamination of isolated swine skin contaminated with Bacillus anthracis Sterne with the chlorine dioxide product resulted in no viable spores sampled. The toxicity of the chlorine dioxide decontaminant was up to two orders of magnitude less than that of sodium hypochlorite in human skin keratinocyte cultures. In summary, the chlorine dioxide based decontaminant efficiently killed Bacillus anthracis spores in liquid suspension, as well as on isolated swine skin, and was less toxic than sodium hypochlorite in cultures of human skin keratinocytes.

  3. Rapid detection of Bacillus anthracis by γ phage amplification and lateral flow immunochromatography.

    Science.gov (United States)

    Cox, Christopher R; Jensen, Kirk R; Mondesire, Roy R; Voorhees, Kent J

    2015-11-01

    New, rapid point-of-need diagnostic methods for Bacillus anthracis detection can enhance civil and military responses to accidental or deliberate dispersal of anthrax as a biological weapon. Current laboratory-based methods for clinical identification of B. anthracis require 12 to 120h, and are confirmed by plaque assay using the well-characterized γ typing phage, which requires an additional minimum of 24h for bacterial culture. To reduce testing time, the natural specificity of γ phage amplification was investigated in combination with lateral flow immunochromatography (LFI) for rapid, point-of-need B. anthracis detection. Phage-based LFI detection of B. anthracis Sterne was validated over a range of bacterial and phage concentrations with optimal detection achieved in as little as 2h from the onset of amplification with a threshold sensitivity of 2.5×10(4)cfu/mL. The novel use of γ phage amplification detected with a simple, inexpensive LFI assay provides a rapid, sensitive, highly accurate, and field-deployable method for diagnostic ID of B. anthracis in a fraction of the time required by conventional techniques, and without the need for extensive laboratory culture.

  4. Growth characteristics of Bacillus anthracis compared to other Bacillus spp. on the selective nutrient media Anthrax Blood Agar and Cereus Ident Agar.

    Science.gov (United States)

    Tomaso, Herbert; Bartling, Carsten; Al Dahouk, Sascha; Hagen, Ralf M; Scholz, Holger C; Beyer, Wolfgang; Neubauer, Heinrich

    2006-01-01

    Anthrax Blood Agar (ABA) and Cereus Ident Agar (CEI) were evaluated as selective growth media for the isolation of Bacillus anthracis using 92 B. anthracis and 132 other Bacillus strains from 30 species. The positive predictive values for the identification of B. anthracis on ABA, CEI, and the combination of both were 72%, 71%, and 90%, respectively. Thus, less than 10% of all species were misidentified using both nutrient media. Species which might be misidentified as B. anthracis were B. cereus, B. mycoides, and B. thuringiensis. Particularly, 30% of B. weihenstephanensis strains were misidentified as B. anthracis.

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

    Directory of Open Access Journals (Sweden)

    Atanu Sengupta

    2012-01-01

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

  6. Structure of 5-formyltetrahydrofolate cyclo-ligase from Bacillus anthracis (BA4489)

    Energy Technology Data Exchange (ETDEWEB)

    Meier, Christoph [Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Carter, Lester G. [Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Winter, Graeme [CCLRC Daresbury Laboratory, Warrington, Cheshire WA4 4AD (United Kingdom); Owens, Ray J. [Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Stuart, David I.; Esnouf, Robert M., E-mail: robert@strubi.ox.ac.uk [Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

    2007-03-01

    The structure of 5-formyltetrahydrofolate cyclo-ligase from B. anthracis determined by X-ray crystallography at a resolution of 1.6 Å is described. Bacillus anthracis is a spore-forming bacterium and the causative agent of the disease anthrax. The Oxford Protein Production Facility has been targeting proteins from B. anthracis in order to develop high-throughput technologies within the Structural Proteomics in Europe project. As part of this work, the structure of 5-formyltetrahydrofolate cyclo-ligase (BA4489) has been determined by X-ray crystallography to 1.6 Å resolution. The structure, solved in complex with magnesium-ion-bound ADP and phosphate, gives a detailed picture of the proposed catalytic mechanism of the enzyme. Chemical differences from other cyclo-ligase structures close to the active site that could be exploited to design specific inhibitors are also highlighted.

  7. Microbial forensics: fiber optic microarray subtyping of Bacillus anthracis

    Science.gov (United States)

    Shepard, Jason R. E.

    2009-05-01

    The past decade has seen increased development and subsequent adoption of rapid molecular techniques involving DNA analysis for detection of pathogenic microorganisms, also termed microbial forensics. The continued accumulation of microbial sequence information in genomic databases now better positions the field of high-throughput DNA analysis to proceed in a more manageable fashion. The potential to build off of these databases exists as technology continues to develop, which will enable more rapid, cost effective analyses. This wealth of genetic information, along with new technologies, has the potential to better address some of the current problems and solve the key issues involved in DNA analysis of pathogenic microorganisms. To this end, a high density fiber optic microarray has been employed, housing numerous DNA sequences simultaneously for detection of various pathogenic microorganisms, including Bacillus anthracis, among others. Each organism is analyzed with multiple sequences and can be sub-typed against other closely related organisms. For public health labs, real-time PCR methods have been developed as an initial preliminary screen, but culture and growth are still considered the gold standard. Technologies employing higher throughput than these standard methods are better suited to capitalize on the limitless potential garnered from the sequence information. Microarray analyses are one such format positioned to exploit this potential, and our array platform is reusable, allowing repetitive tests on a single array, providing an increase in throughput and decrease in cost, along with a certainty of detection, down to the individual strain level.

  8. Transient lipopolysaccharide-induced resistance to aerosolized Bacillus anthracis in New Zealand white rabbits.

    Science.gov (United States)

    Yee, Steven B; Dyer, David N; Twenhafel, Nancy A; Pitt, M Louise M

    2013-06-01

    Previous studies have demonstrated that prior infection by various bacterial pathogens induces nonspecific resistance to subsequent infection by other gram-negative and gram-positive bacterial pathogens. In the present study, we evaluated whether underlying inflammation enhanced host resistance to inhalational Bacillus anthracis infection in New Zealand White rabbits (SPF; Bordetella- and Pasteurella-free). Accordingly, rabbits were pretreated with either the inflammagen bacterial LPS (60,000 EU/kg), a component of the outer membrane of gram-negative bacteria, or saline (vehicle). Administration of LPS resulted in brief pyrexia and a significant increase in the proinflammatory cytokine TNFα, thus confirming LPS-induced inflammation. At 24 h after LPS treatment, rabbits were exposed to aerosolized B. anthracis spores (Ames strain; approximately 300 LD50). Blood samples collected at various times after challenge were cultured. Compared with their saline-pretreated counterparts, LPS-pretreated, B. anthracis challenged rabbits exhibited delays in 2 biomarkers of B. anthracis infection-anthrax-induced pyrexia (25 h versus 66 h after challenge, respectively) and bacteremia (26 h versus 63 h, respectively)-and survived longer (41 h versus 90 h, respectively). Similar to control animals, all LPS-pretreated, B. anthracis-challenged rabbits exhibited pathology consistent with inhalational anthrax. Taken together, these results suggest that prior or underlying stimulation of the innate immune system induces transient host resistance to subsequent B. anthracis infection in SPF New Zealand white rabbits. In particular, our results emphasize the importance of using animals that are free of underlying infections to prevent confounding data in studies for inhalational anthrax characterization and medical countermeasure evaluation.

  9. Alveolar macrophages infected with Ames or Sterne strain of Bacillus anthracis elicit differential molecular expression patterns.

    Directory of Open Access Journals (Sweden)

    Felicia D Langel

    Full Text Available Alveolar macrophages (AMs phagocytose Bacillus anthracis following inhalation and induce the production of pro-inflammatory cytokines and chemokines to mediate the activation of innate immunity. Ames, the virulent strain of B. anthracis, contains two plasmids that encode the antiphagocytic poly-γ-d-glutamic acid capsule and the lethal toxin. The attenuated Sterne strain of B. anthracis, which lacks the plasmid encoding capsule, is widely adapted as a vaccine strain. Although differences in the outcome of infection with the two strains may have originated from the presence or absence of an anti-phagocytic capsule, the disease pathogenesis following infection will be manifested via the host responses, which is not well understood. To gain understanding of the host responses at cellular level, a microarray analysis was performed using primary rhesus macaque AMs infected with either Ames or Sterne spores. Notably, 528 human orthologs were identified to be differentially expressed in AMs infected with either strain of the B. anthracis. Meta-analyses revealed genes differentially expressed in response to B. anthracis infection were also induced upon infections with multiple pathogens such as Francisella Novicida or Staphylococcus aureus. This suggests the existence of a common molecular signature in response to pathogen infections. Importantly, the microarray and protein expression data for certain cytokines, chemokines and host factors provide further insights on how cellular processes such as innate immune sensing pathways, anti-apoptosis versus apoptosis may be differentially modulated in response to the virulent or vaccine strain of B. anthracis. The reported differences may account for the marked difference in pathogenicity between these two strains.

  10. Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group.

    Science.gov (United States)

    Ahmod, Nadia Z; Gupta, Radhey S; Shah, Haroun N

    2011-12-01

    Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have identified a 24 amino acid deletion in a conserved region of the YeaC protein that is uniquely present in B. anthracis. PCR primers based on conserved regions flanking this indel in the Bacillus cereus group of species (viz. Bacillus cereus, B. anthracis, B. thuringiensis, B. mycoides, B. weihenstephnensis and B. pseudomycoides) specifically amplified a 282 bp fragment from all six reference B. anthracis strains, whereas a 354 bp fragment was amplified from 15 other B. cereus group of species/strains. These fragments, due to large size difference, are readily distinguished by means of agarose gel electrophoresis. In contrast to the B. cereus group, no PCR amplification was observed with any of the non-B. cereus group of species/strains. This indel was also used for developing a rapid pyrosequencing assay for the identification of B. anthracis. Its performance was evaluated by examining the presence or absence of this indel in a panel of 81 B. cereus-like isolates from various sources that included 39 B. anthracis strains. Based upon the sequence data from the pyrograms, the yeaC indel was found to be a distinctive characteristic of various B. anthracis strains tested and not found in any other species/strains from these samples. Therefore, this B. anthracis specific indel provides a robust and highly-specific chromosomal marker for the identification of this high-risk pathogen from other members of the B. cereus group independent of a strain's virulence. The pyrosequencing platform also allows for the rapid and simultaneous screening of multiple samples for the presence of this B. anthracis-specific marker.

  11. Effects of endogenous D-alanine synthesis and autoinhibition of Bacillus anthracis germination on in vitro and in vivo infections.

    Science.gov (United States)

    McKevitt, Matthew T; Bryant, Katie M; Shakir, Salika M; Larabee, Jason L; Blanke, Steven R; Lovchik, Julie; Lyons, C Rick; Ballard, Jimmy D

    2007-12-01

    Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous D-alanine production to the efficiency and timing of B. anthracis spore germination under in vitro and in vivo conditions. Racemase-mediated production of endogenous D-alanine by B. anthracis altered the kinetics for initiation of germination over a range of spore densities and exhibited a threshold effect wherein small changes in spore number resulted in major changes in germination efficiency. This threshold effect correlated with D-alanine production, was prevented by an alanine racemase inhibitor, and required L-alanine. Interestingly, endogenous production of inhibitory levels of D-alanine was detected under experimental conditions that did not support germination and in a germination-deficient mutant of B. anthracis. Racemase-dependent production of D-alanine enhanced survival of B. anthracis during interaction with murine macrophages, suggesting a role for inhibition of germination during interaction with these cells. Finally, in vivo experiments revealed an approximately twofold decrease in the 50% lethal dose of B. anthracis spores administered in the presence of D-alanine, indicating that rates of germination may be directly influenced by the levels of this amino acid during early stages of disease.

  12. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    OpenAIRE

    Ågren, Joakim; Raditijo A Hamidjaja; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; van Rotterdam, Bart; Derzelle, Sylviane

    2013-01-01

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. ...

  13. 76 FR 53480 - Prospective Grant of Exclusive License: Conjugate Vaccines Against B. anthracis

    Science.gov (United States)

    2011-08-26

    ... HUMAN SERVICES National Institutes of Health Prospective Grant of Exclusive License: Conjugate Vaccines... factor'' (EF). Although production of an efficient anthrax vaccine is an ultimate goal, the benefits of... therapy of B. anthracis (anthrax) infection by immunization with conjugate vaccines against anthrax...

  14. Crossing of the epithelial barriers by Bacillus anthracis: the Known and the Unknown.

    Science.gov (United States)

    Goossens, Pierre L; Tournier, Jean-Nicolas

    2015-01-01

    Anthrax, caused by Bacillus anthracis, a Gram-positive spore-forming bacterium, is initiated by the entry of spores into the host body. There are three types of human infection: cutaneous, inhalational, and gastrointestinal. For each form, B. anthracis spores need to cross the cutaneous, respiratory or digestive epithelial barriers, respectively, as a first obligate step to establish infection. Anthrax is a toxi-infection: an association of toxemia and rapidly spreading infection progressing to septicemia. The pathogenicity of Bacillus anthracis mainly depends on two toxins and a capsule. The capsule protects bacilli from the immune system, thus promoting systemic dissemination. The toxins alter host cell signaling, thereby paralyzing the immune response of the host and perturbing the endocrine and endothelial systems. In this review, we will mainly focus on the events and mechanisms leading to crossing of the respiratory epithelial barrier, as the majority of studies have addressed inhalational infection. We will discuss the critical gaps of knowledge that need to be addressed to gain a comprehensive view of the initial steps of inhalational anthrax. We will then discuss the few data available on B. anthracis crossing the cutaneous and digestive epithelia.

  15. Crossing of the epithelial barriers by Bacillus anthracis: the Known and the Unknown

    Directory of Open Access Journals (Sweden)

    Pierre L Goossens

    2015-10-01

    Full Text Available Anthrax, caused by Bacillus anthracis, a Gram-positive spore-forming bacterium, is initiated by the entry of spores into the host body. There are three types of human infection: cutaneous, inhalational, and gastrointestinal. For each form, B. anthracis spores need to cross the cutaneous, respiratory or digestive epithelial barriers, respectively, as a first obligate step to establish infection. Anthrax is a toxi-infection: an association of toxemia and rapidly spreading infection progressing to septicemia. The pathogenicity of Bacillus anthracis mainly depends on two toxins and a capsule. The capsule protects bacilli from the immune system, thus promoting systemic dissemination. The toxins alter host cell signalling, thereby paralysing the immune response of the host and perturbing the endocrine and endothelial systems.In this review, we will mainly focus on the events and mechanisms leading to crossing of the respiratory epithelial barrier, as the majority of studies have addressed inhalational infection. We will discuss the critical gaps of knowledge that need to be addressed to gain a comprehensive view of the initial steps of inhalational anthrax. We will then discuss the few data available on B. anthracis crossing the cutaneous and digestive epithelia.

  16. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    Directory of Open Access Journals (Sweden)

    Ma’ayan Israeli

    2016-08-01

    Full Text Available Edema Factor (EF, the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP, and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules.

  17. Identification and characterization of Bacillus anthracis by multiplex PCR on DNA chip.

    Science.gov (United States)

    Wang, Shi-Hua; Wen, Ji-Kai; Zhou, Ya-Feng; Zhang, Zhi-Ping; Yang, Rui-Fu; Zhang, Ji-Bin; Chen, Jia; Zhang, Xian-En

    2004-11-01

    Bacillus anthracis can be identified by detecting virulence factor genes located on two plasmids, pXO1 and pXO2. Combining multiplex PCR with arrayed anchored primer PCR and biotin-avidin alkaline phosphatase indicator system, we developed a qualitative DNA chip method for characterization of B. anthracis, and simultaneous confirmation of the species identity independent of plasmid contents. The assay amplifies pag gene (in pXO1), cap gene (in pXO2) and Ba813 gene (a B. anthracis specific chromosomal marker), and the results were indicated by an easy-to-read profile based on the color reaction of alkaline phosphatase. About 1 pg of specific DNA fragments on the chip wells could be detected after PCR. With the proposed method, the avirulent (pXO1+/2-, pXO1-/2+ and pXO1-/2-) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria were unambiguously identified, while the genera other than Bacillus gave no positive signal.

  18. The Effect of Growth Medium on B. anthracis Sterne Spore Carbohydrate Content

    Energy Technology Data Exchange (ETDEWEB)

    Colburn, Heather A.; Wunschel, David S.; Antolick, Kathryn C.; Melville, Angela M.; Valentine, Nancy B.

    2011-06-01

    The expressed characteristics of biothreat agents may be impacted by variations in the culture environment, including growth medium formulation. The carbohydrate composition of B. anthracis spores has been well studied, particularly for the exosporium, which is the outermost spore structure. The carbohydrate composition of the exosporium has been demonstrated to be distinct from the vegetative form containing unique monosaccharides.

  19. clpC operon regulates cell architecture and sporulation in Bacillus anthracis.

    Science.gov (United States)

    Singh, Lalit K; Dhasmana, Neha; Sajid, Andaleeb; Kumar, Prasun; Bhaduri, Asani; Bharadwaj, Mitasha; Gandotra, Sheetal; Kalia, Vipin C; Das, Taposh K; Goel, Ajay K; Pomerantsev, Andrei P; Misra, Richa; Gerth, Ulf; Leppla, Stephen H; Singh, Yogendra

    2015-03-01

    The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knockout strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and open up further interest on this operon, which might be of importance to success of B. anthracis as pathogen.

  20. Functional characterization of WalRK: A two-component signal transduction system from Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Alisha Dhiman

    2014-01-01

    Full Text Available Two-component signal transduction systems (TCS, consisting of a sensor histidine protein kinase and its cognate response regulator, are an important mode of environmental sensing in bacteria. Additionally, they have been found to regulate virulence determinants in several pathogens. Bacillus anthracis, the causative agent of anthrax and a bioterrorism agent, harbours 41 pairs of TCS. However, their role in its pathogenicity has remained largely unexplored. Here, we show that WalRK of B. anthracis forms a functional TCS which exhibits some species-specific functions. Biochemical studies showed that domain variants of WalK, the histidine kinase, exhibit classical properties of autophosphorylation and phosphotransfer to its cognate response regulator WalR. Interestingly, these domain variants also show phosphatase activity towards phosphorylated WalR, thereby making WalK a bifunctional histidine kinase/phosphatase. An in silico regulon determination approach, using a consensus binding sequence from Bacillus subtilis, provided a list of 30 genes that could form a putative WalR regulon in B. anthracis. Further, electrophoretic mobility shift assay was used to show direct binding of purified WalR to the upstream regions of three putative regulon candidates, an S-layer protein EA1, a cell division ABC transporter FtsE and a sporulation histidine kinase KinB3. Our work lends insight into the species-specific functions and mode of action of B. anthracis WalRK.

  1. Bacillus anthracis diagnostic detection and rapid antibiotic susceptibility determination using 'bioluminescent' reporter phage.

    Science.gov (United States)

    Schofield, David A; Sharp, Natasha J; Vandamm, Joshua; Molineux, Ian J; Spreng, Krista A; Rajanna, Chythanya; Westwater, Caroline; Stewart, George C

    2013-11-01

    Genetically modified phages have the potential to detect pathogenic bacteria from clinical, environmental, or food-related sources. Herein we assess an engineered 'bioluminescent' reporter phage (Wß::luxAB) as a clinical diagnostic tool for Bacillus anthracis, the etiological agent of anthrax. Wß::luxAB is able to rapidly (within minutes) detect a panel of B. anthracis strains by transducing a bioluminescent phenotype. The reporter phage displays species specificity by its inability, or significantly reduced ability, to detect members of the closely related Bacillus cereus group and other common bacterial pathogens. Using spiked clinical specimens, Wß::luxAB detects B. anthracis within 5 h at clinically relevant concentrations, and provides antibiotic susceptibility information that mirrors the CLSI method, except that data are obtained at least 5-fold faster. Although anthrax is a treatable disease, a positive patient prognosis is dependent on timely diagnosis and appropriate therapy. Wß::luxAB rapidly detects B. anthracis and determines antibiotic efficacy, properties that will help patient outcome.

  2. Identification of Bacillus anthracis PurE inhibitors with antimicrobial activity.

    Science.gov (United States)

    Kim, Anna; Wolf, Nina M; Zhu, Tian; Johnson, Michael E; Deng, Jiangping; Cook, James L; Fung, Leslie W-M

    2015-04-01

    N(5)-carboxy-amino-imidazole ribonucleotide (N(5)-CAIR) mutase (PurE), a bacterial enzyme in the de novo purine biosynthetic pathway, has been suggested to be a target for antimicrobial agent development. We have optimized a thermal shift method for high-throughput screening of compounds binding to Bacillus anthracis PurE. We used a low ionic strength buffer condition to accentuate the thermal shift stabilization induced by compound binding to Bacillus anthracis PurE. The compounds identified were then subjected to computational docking to the active site to further select compounds likely to be inhibitors. A UV-based enzymatic activity assay was then used to select inhibitory compounds. Minimum inhibitory concentration (MIC) values were subsequently obtained for the inhibitory compounds against Bacillus anthracis (ΔANR strain), Escherichia coli (BW25113 strain, wild-type and ΔTolC), Francisella tularensis, Staphylococcus aureus (both methicillin susceptible and methicillin-resistant strains) and Yersinia pestis. Several compounds exhibited excellent (0.05-0.15μg/mL) MIC values against Bacillus anthracis. A common core structure was identified for the compounds exhibiting low MIC values. The difference in concentrations for inhibition and MIC suggest that another enzyme(s) is also targeted by the compounds that we identified.

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

    Science.gov (United States)

    Cote, Christopher K; Welkos, Susan L

    2015-08-17

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

  4. 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 ...

  5. The two CcdA proteins of Bacillus anthracis differentially affect virulence gene expression and sporulation.

    Science.gov (United States)

    Han, Hesong; Wilson, Adam C

    2013-12-01

    The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation, while the closely related organism Bacillus subtilis carries only one copy of ccdA. To investigate the roles of the two ccdA gene copies in B. anthracis, strains were constructed without each ccdA gene, and one strain was constructed without both copies simultaneously. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. Complementation and expression analyses indicate that ccdA2 encodes the primary CcdA in B. anthracis, active in all three pathways. While CcdA1 retains activity in cytochrome c maturation and virulence control, it has completely lost its activity in the sporulation pathway. In support of this finding, expression of ccdA1 is strongly reduced when cells are grown under sporulation-inducing conditions. When the activities of CcdA1 and CcdA2 were analyzed in B. subtilis, neither protein retained activity in cytochrome c maturation, but CcdA2 could still function in sporulation. These observations reveal the complexities of thiol-disulfide oxidoreductase function in pathways relevant to virulence and physiology.

  6. BAC Library Construction and Physical Mapping of Bacillus anthracis A16R

    Institute of Scientific and Technical Information of China (English)

    Zhang Da; Zhu Houchu; Huang Liuyu

    2013-01-01

    Bacillus anthracis is an endospore-forming bacterium that causes severe inhalational anthrax, and bacillus anthracis A16R is an attenuated strain derived from Bacillus anthracis A16. The development of bacterial artificial chromosome (BAC) system has allowed the construction of large insert-size DNA libraries, and the bacterial artificial chromosomes (BACs) have become the preferred large insert cloning system for genomic analysis because such libraries are characteristically stable, high in ifdelity and easy to handle. To facilitate genome studies of this bacterium, a bacterial artiifcial chromosome library (BAC) has been established from genome DNA of Bacillus anthracis A16R. This library consisted of 9 600 clones randomly selected from more than 15 000 recombinant clones carrying inserts in the plindigoBAC-5 vectors. The mean insert size was 56 kbp, representing an approximate 12-fold genome coverage, while end sequences were obtained from 700 randomly selected clones. Sequences were compared with Bacillus anthracis Ames and Bacillus cereus ATCC 14579 Genome Project databases using the NCBI BLASTN search project. And most BLASTN results showed high identities and that the sequences’ sites could be used as STSs. To construct this physical map, Excel was used for the array of STSs and some gaps of the map were iflled up by PCR walking. Artemis-V4 was used in the construction of a genome-wide physical map with 93%genome coverage. The A16R BAC library proved to be a vital tool for the generation of a map that would not only allow the subsequent sequencing of defined areas of genome, but also provide immediate access to clones that were stable and convenient for functional genomic researches.

  7. 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.

  8. The Bacillus anthracis cholesterol-dependent cytolysin, Anthrolysin O, kills human neutrophils, monocytes and macrophages

    Directory of Open Access Journals (Sweden)

    Rest Richard F

    2006-06-01

    Full Text Available Abstract Background Bacillus anthracis is an animal and human pathogen whose virulence is characterized by lethal and edema toxin, as well as a poly-glutamic acid capsule. In addition to these well characterized toxins, B. anthracis secretes several proteases and phospholipases, and a newly described toxin of the cholesterol-dependent cytolysin (CDC family, Anthrolysin O (ALO. Results In the present studies we show that recombinant ALO (rALO or native ALO, secreted by viable B. anthracis, is lethal to human primary polymorphonuclear leukocytes (PMNs, monocytes, monocyte-derived macrophages (MDMs, lymphocytes, THP-1 monocytic human cell line and ME-180, Detroit 562, and A549 epithelial cells by trypan blue exclusion or lactate dehydrogenase (LDH release viability assays. ALO cytotoxicity is dose and time dependent and susceptibility to ALO-mediated lysis differs between cell types. In addition, the viability of monocytes and hMDMs was assayed in the presence of vegetative Sterne strains 7702 (ALO+, UT231 (ALO-, and a complemented strain expressing ALO, UT231 (pUTE544, and was dependent upon the expression of ALO. Cytotoxicity of rALO is seen as low as 0.070 nM in the absence of serum. All direct cytotoxic activity is inhibited by the addition of cholesterol or serum concentration as low as 10%. Conclusion The lethality of rALO and native ALO on human monocytes, neutrophils, macrophages and lymphocytes supports the idea that ALO may represent a previously unidentified virulence factor of B. anthracis. The study of other factors produced by B. anthracis, along with the major anthrax toxins, will lead to a better understanding of this bacterium's pathogenesis, as well as provide information for the development of antitoxin vaccines for treating and preventing anthrax.

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

    Science.gov (United States)

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

    2016-01-01

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

  10. BAC Library Construction and Physical Mapping of Bacillus anthracis A16R

    Directory of Open Access Journals (Sweden)

    Da Zhang

    2013-12-01

    Full Text Available Bacillus anthracis is an endospore-forming bacterium that causes severe inhalational anthrax, and bacillus anthracis A16R is an attenuated strain derived from Bacillus anthracis A16. The development of bacterial artificial chromosome (BAC system has allowed the construction of large insert-size DNA libraries, and the bacterial artificial chromosomes (BACs have become the preferred large insert cloning system for genomic analysis because such libraries are characteristically stable, high in fidelity and easy to handle. To facilitate genome studies of this bacterium, a bacterial artificial chromosome library (BAC has been established from genome DNA of Bacillus anthracis A16R. This library consisted of 9 600 clones randomly selected from more than 15 000 recombinant clones carrying inserts in the plindigoBAC-5 vectors. The mean insert size was 56 kbp, representing an approximate 12-fold genome coverage, while end sequences were obtained from 700 randomly selected clones. Sequences were compared with Bacillus anthracis Ames and Bacillus cereus ATCC 14579 Genome Project databases using the NCBI BLASTN search project. And most BLASTN results showed high identities and that the sequences’ sites could be used as STSs. To construct this physical map, Excel was used for the array of STSs and some gaps of the map were filled up by PCR walking. Artemis-V4 was used in the construction of a genome-wide physical map with 93% genome coverage. The A16R BAC library proved to be a vital tool for the generation of a map that would not only allow the subsequent sequencing of defined areas of genome, but also provide immediate access to clones that were stable and convenient for functional genomic researches.

  11. The Pathogenomic Sequence Analysis of B. cereus and B.thuringiensis Isolates Closely Related to Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Han, Cliff S.; Xie, Gary; Challacombe, Jean F.; Altherr, MichaelR.; Smriti, B.; Bruce, David; Campbell, Connie S.; Campbell, Mary L.; Chen, Jin; Chertkov, Olga; Cleland, Cathy; Dimitrijevic-Bussod, M.; Doggett, Norman A.; Fawcett, John J.; Glavina, Tijana; Goodwin, Lynne A.; 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 P.; Richardson, Paul; Robinson, DonnaL.; Rubin, Eddy; Saunders, Elizabeth; Tapia, Roxanne; Tesmer, Judith G.; Thayer, Nina; Thompson, Linda S.; Tice, Hope; Ticknor, Lawrence O.; Wills, Patti L.; Gilna, Payl; Brettin, Thomas S.

    2005-08-18

    The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B.cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including Banthracis. Comparative analysis of these two genomes with other members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.

  12. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    DEFF Research Database (Denmark)

    Ågren, Joakim; Hamidjaja, Raditijo A.; Hansen, Trine

    2013-01-01

    on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S......Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely......-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal...

  13. Microarray Analysis of Transposon Insertion Mutants in Bacillus Anthracis: Global Identification of Genes Required for Sporulation and Germination

    Science.gov (United States)

    2007-02-01

    Gilois, M. Rose, and D. Lereclus. 2001. Oligopep- tide permease is required for expression of the Bacillus thuringiensis plcR regulon and for...non- toxin gene expression in Bacillus anthracis. Infect. Immun. 65:3091–3099. 10. Ikeda, R. A., C. M. Ligman, and S. Warshamana. 1992. T7 promoter con...nontoxi- genic Bacillus anthracis spore vaccines based on strains expressing mutant vari- ants of lethal toxin components. Vaccine 23:5688–5697. 17. Read

  14. Characterization of Bacillus anthracis-like bacteria isolated from wild great apes from Cote d'Ivoire and Cameroon.

    Science.gov (United States)

    Klee, Silke R; Ozel, Muhsin; Appel, Bernd; Boesch, Christophe; Ellerbrok, Heinz; Jacob, Daniela; Holland, Gudrun; Leendertz, Fabian H; Pauli, Georg; Grunow, Roland; Nattermann, Herbert

    2006-08-01

    We present the microbiological and molecular characterization of bacteria isolated from four chimpanzees and one gorilla thought to have died of an anthrax-like disease in Côte d'Ivoire and Cameroon. These isolates differed significantly from classic Bacillus anthracis by the following criteria: motility, resistance to the gamma phage, and, for isolates from Cameroon, resistance to penicillin G. A capsule was expressed not only after induction by CO(2) and bicarbonate but also under normal growth conditions. Subcultivation resulted in beta-hemolytic activity and gamma phage susceptibility in some subclones, suggesting differences in gene regulation compared to classic B. anthracis. The isolates from Côte d'Ivoire and Cameroon showed slight differences in their biochemical characteristics and MICs of different antibiotics but were identical in all molecular features and sequences analyzed. PCR and Southern blot analyses confirmed the presence of both the toxin and the capsule plasmid, with sizes corresponding to the B. anthracis virulence plasmids pXO1 and pXO2. Protective antigen was expressed and secreted into the culture supernatant. The isolates possessed variants of the Ba813 marker and the SG-749 fragment differing from that of classic B. anthracis strains. Multilocus sequence typing revealed a close relationship of our atypical isolates with both classic B. anthracis strains and two uncommonly virulent Bacillus cereus and Bacillus thuringiensis isolates. We propose that the newly discovered atypical B. anthracis strains share a common ancestor with classic B. anthracis or that they emerged recently by transfer of the B. anthracis plasmids to a strain of the B. cereus group.

  15. A new approach to in silico SNP detection and some new SNPs in the Bacillus anthracis genome

    OpenAIRE

    Francoeur Joe; Brodzik Andrzej K

    2011-01-01

    Abstract Background Bacillus anthracis is one of the most monomorphic pathogens known. Identification of polymorphisms in its genome is essential for taxonomic classification, for determination of recent evolutionary changes, and for evaluation of pathogenic potency. Findings In this work three strains of the Bacillus anthracis genome are compared and previously unpublished single nucleotide polymorphisms (SNPs) are revealed. Moreover, it is shown that, despite the highly monomorphic nature o...

  16. Discerning Viable from Nonviable Yersinia pestis pgm- and Bacillus anthracis Sterne using Propidium Monoazide in the Presence of White Powders

    Energy Technology Data Exchange (ETDEWEB)

    Hess, Becky M.; Kaiser, Brooke LD; Sydor, Michael A.; Wunschel, David S.; Bruckner-Lea, Cindy J.; Hutchison, Janine R.

    2015-12-23

    ABSTRACT Aims To develop and optimize an assay to determine viability status of Bacillus anthracis Sterne and Yersinia pestis pgm- strains in the presence of white powders by coupling propidium monoazide (PMA) treatment with real-time PCR (qPCR) analysis. Methods and Results PMA selectively enters nonviable cells and binds DNA, thereby increasing qPCR assay cycle threshold (CT) values compared to untreated samples. Dye concentration, cell number and fitness, incubation time, inactivation methods, and assay buffer were optimized for B. anthracis Sterne and Y. pestis pgm-. Differences in CT values in nonviable cells compared to untreated samples were consistently > 9 for both B. anthracis Sterne vegetative cells and Y. pestis pgm- in the presence and absence of three different white powders. Our method eliminates the need for a DNA extraction step prior to detection by qPCR. Conclusions The developed assay enables simultaneous identification and viability assessment for B. anthracis Sterne and Y. pestis pgm- under laboratory conditions, even in the presence of white powders. Eliminating the DNA extraction step that is typically used reduces total assay time and labor requirements for sample analysis. Significance and Impact of the Study The method developed for simultaneous detection and viability assessment for B. anthracis and Y. pestis can be employed in forming decisions about the severity of a biothreat event or the safety of food. Keywords Bacillus anthracis, Yersinia pestis, Propidium Monoazide, qPCR, White Powders, Rapid Viability Detection

  17. Identification and Classification of bcl Genes and Proteins of Bacillus cereus Group Organisms and Their Application in Bacillus anthracis Detection and Fingerprinting▿ †

    OpenAIRE

    Leski, Tomasz A.; Caswell, Clayton C.; Pawlowski, Marcin; Klinke, David J.; Bujnicki, Janusz M.; Hart, Sean J.; Lukomski, Slawomir

    2009-01-01

    The Bacillus cereus group includes three closely related species, B. anthracis, B. cereus, and B. thuringiensis, which form a highly homogeneous subdivision of the genus Bacillus. One of these species, B. anthracis, has been identified as one of the most probable bacterial biowarfare agents. Here, we evaluate the sequence and length polymorphisms of the Bacillus collagen-like protein bcl genes as a basis for B. anthracis detection and fingerprinting. Five genes, designated bclA to bclE, are p...

  18. Occurrence and Genetic Diversity of Bacillus anthracis Strains Isolated in an Active Wool-Cleaning Factory▿

    Science.gov (United States)

    Wattiau, Pierre; Klee, Silke R.; Fretin, David; Van Hessche, Mieke; Ménart, Marie; Franz, Tatjana; Chasseur, Camille; Butaye, Patrick; Imberechts, Hein

    2008-01-01

    Culturable microorganisms from various samples taken at an active factory performing wool and goat hair cleaning were isolated and analyzed. Bacillus anthracis was found in air filter dust, wastewater, and goat hairs, where it accounted for approximately 1% of the total counts of viable bacteria. Consistent with the countries of origin of the processed material (South Caucasian and Middle Eastern), all B. anthracis isolates belonged to the same phylogenetic cluster, as determined by variable-number tandem repeat (VNTR) typing at eight loci. Within this cluster, five closely related VNTR subtypes could be identified, of which two were previously unreported. Additional diversity was observed when more sensitive genetic markers were assayed, demonstrating the multifocal nature of goat hair contamination. Goat hair originating from areas where anthrax is endemic remains a material with high biological risk for modern woolworkers. PMID:18487406

  19. Effects of Long-Term Storage on Plasmid Stability in Bacillus anthracis

    Science.gov (United States)

    Marston, Chung K.; Hoffmaster, Alex R.; Wilson, Kathy E.; Bragg, Sandra L.; Plikaytis, Brian; Brachman, Philip; Johnson, Scott; Kaufmann, Arnold F.; Popovic, Tanja

    2005-01-01

    The plasmid profiles of 619 cultures of Bacillus anthracis which had been isolated and stored between 1954 and 1989 were analyzed using the Laboratory Response Network real-time PCR assay targeting a chromosomal marker and both virulence plasmids (pXO1 and pXO2). The cultures were stored at ambient temperature on tryptic soy agar slants overlaid with mineral oil. When data were stratified by decade, there was a decreasing linear trend in the proportion of strains containing both plasmids with increased storage time (P < 0.001). There was no significant difference in the proportion of strains containing only pXO1 or strains containing only pXO2 (P = 0.25), but there was a statistical interdependence between the two plasmids (P = 0.004). Loss of viability of B. anthracis cultures stored on agar slants is also discussed. PMID:16332750

  20. Laboratory Studies on Surface Sampling of Bacillus anthracis Contamination: Summary, Gaps, and Recommendations

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F.; Amidan, Brett G.; Hu, Rebecca

    2011-11-28

    This report summarizes previous laboratory studies to characterize the performance of methods for collecting, storing/transporting, processing, and analyzing samples from surfaces contaminated by Bacillus anthracis or related surrogates. The focus is on plate culture and count estimates of surface contamination for swab, wipe, and vacuum samples of porous and nonporous surfaces. Summaries of the previous studies and their results were assessed to identify gaps in information needed as inputs to calculate key parameters critical to risk management in biothreat incidents. One key parameter is the number of samples needed to make characterization or clearance decisions with specified statistical confidence. Other key parameters include the ability to calculate, following contamination incidents, the (1) estimates of Bacillus anthracis contamination, as well as the bias and uncertainties in the estimates, and (2) confidence in characterization and clearance decisions for contaminated or decontaminated buildings. Gaps in knowledge and understanding identified during the summary of the studies are discussed and recommendations are given for future studies.

  1. The Ba813 chromosomal DNA sequence effectively traces the whole Bacillus anthracis community.

    Science.gov (United States)

    Ramisse, V; Patra, G; Vaissaire, J; Mock, M

    1999-08-01

    Plasmid genes that are responsible for virulence of Bacillus anthracis are important targets for the DNA-based detection of anthrax. We evaluated the distribution of the Ba813 chromosomal DNA sequence (Ba813) within closely related Bacillus species. Ba813 was systematically identified from 47 strains or isolates of B. anthracis tested, thus indicating its reliability as a tracer for that species. From the 60 strains of closely related Bacillus spp. examined, three bona fide B. cereus and one bona fide B. thuringiensis were found to harbour Ba813. This marker was also detected in Bacillus sp. isolates that were present at high levels in soil samples collected in a place where an anthrax outbreak had occurred. The significance and the possible function of the Ba813 locus is discussed.

  2. A genetic approach for the identification of exosporium assembly determinants of Bacillus anthracis

    Science.gov (United States)

    Spreng, Krista A.; Thompson, Brian M.; Stewart, George C.

    2013-01-01

    The exosporium is the outermost layer of spores of the zoonotic pathogen Bacillus anthracis. The composition of the exosporium and its functions are only partly understood. Because this outer spore layer is refractive to traditional biochemical analysis, a genetic approach is needed in order to define the proteins which comprise this important spore layer and its assembly pathway. We have created a novel genetic screening system for the identification and isolation of mutants with defects in exosporium assembly during B. anthracis spore maturation. The system is based on the targeting sequence of the BclA exosporium nap layer glycoprotein and a fluorescent reporter. By utilizing this screening system and gene inactivation with Tn916, several novel putative exosporium-associated determinants were identified. A sampling of the mutants obtained was further characterized, confirming their exosporium defect and validating the utility of this screen to identify novel spore determinants in the genome of this pathogen. PMID:23411372

  3. Transcriptional profiling of Bacillus anthracis Sterne (34F2 during iron starvation.

    Directory of Open Access Journals (Sweden)

    Paul E Carlson

    Full Text Available Lack of available iron is one of many environmental challenges that a bacterium encounters during infection and adaptation to iron starvation is important for the pathogen to efficiently replicate within the host. Here we define the transcriptional response of B. anthracis Sterne (34F(2 to iron depleted conditions. Genome-wide transcript analysis showed that B. anthracis undergoes considerable changes in gene expression during growth in iron-depleted media, including the regulation of known and candidate virulence factors. Two genes encoding putative internalin proteins were chosen for further study. Deletion of either gene (GBAA0552 or GBAA1340 resulted in attenuation in a murine model of infection. This attenuation was amplified in a double mutant strain. These data define the transcriptional changes induced during growth in low iron conditions and illustrate the potential of this dataset in the identification of putative virulence determinants for future study.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-25

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

  5. Specific activation of dendritic cells enhances clearance of Bacillus anthracis following infection.

    Science.gov (United States)

    Thompson, Iain J T; Mann, Elizabeth R; Stokes, Margaret G; English, Nicholas R; Knight, Stella C; Williamson, Diane

    2014-01-01

    Dendritic cells are potent activators of the immune system and have a key role in linking innate and adaptive immune responses. In the current study we have used ex vivo pulsed bone marrow dendritic cells (BMDC) in a novel adoptive transfer strategy to protect against challenge with Bacillus anthracis, in a murine model. Pre-pulsing murine BMDC with either recombinant Protective Antigen (PA) or CpG significantly upregulated expression of the activation markers CD40, CD80, CD86 and MHC-II. Passive transfusion of mice with pulsed BMDC, concurrently with active immunisation with rPA in alum, significantly enhanced (pBMDC, demonstrated 100% survival following lethal B. anthracis challenge and had significantly enhanced (p<0.05) bacterial clearance within 2 days, compared with mice vaccinated with rPA and alum alone.

  6. Differentiation of Bacillus anthracis from Bacillus cereus by gas chromatographic whole-cell fatty acid analysis.

    OpenAIRE

    Lawrence, D.; Heitefuss, S; Seifert, H S

    1991-01-01

    Three strains of Bacillus anthracis and seven strains of Bacillus cereus were grown on complex medium and on synthetic medium. Gas chromatographic analysis of whole-cell fatty acids of strains grown on complex medium gave nearly identical fatty acid patterns. Fatty acid patterns of strains grown on synthetic medium showed a high content of branched-chain fatty acids. Significant differences between the fatty acid patterns of the two species were found. Odd iso/anteiso fatty acid ratios were a...

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

    OpenAIRE

    2014-01-01

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

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

    OpenAIRE

    2014-01-01

    The infectious agent of the disease anthrax is the spore of Bacillus anthracis. Bacterial spores are extremely resistant to environmental stresses, which greatly hinders spore decontamination efforts. The spore cortex, a thick layer of modified peptidoglycan, contributes to spore dormancy and resistance by maintaining the low water content of the spore core. The cortex is degraded by germination-specific lytic enzymes (GSLEs) during spore germination, rendering the cells vulnerable to common ...

  9. Anthrax, Toxins and Vaccines: A 125-Year Journey Targeting Bacillus anthracis

    Science.gov (United States)

    2009-01-01

    after their inception. Since these early begin- nings, additional medical history linked to B. anthracis coincides in many ways with vac- cines that...by vaccination. Hopefully, the reader will be challenged to think of how existing anthrax vaccines, especially those meant for humans, can be...improved with available knowledge/technology. The many current anthrax vaccines are linked to Louis Pasteur’s seminal experiments at Pouilly Ie-Fort in

  10. A Bacillus anthracis Genome Sequence from the Sverdlovsk 1979 Autopsy Specimens

    Science.gov (United States)

    Sahl, Jason W.; Pearson, Talima; Okinaka, Richard; Schupp, James M.; Gillece, John D.; Heaton, Hannah; Birdsell, Dawn; Hepp, Crystal; Fofanov, Viacheslav; Noseda, Ramón; Fasanella, Antonio; Hoffmaster, Alex; Wagner, David M.

    2016-01-01

    ABSTRACT Anthrax is a zoonotic disease that occurs naturally in wild and domestic animals but has been used by both state-sponsored programs and terrorists as a biological weapon. A Soviet industrial production facility in Sverdlovsk, USSR, proved deficient in 1979 when a plume of spores was accidentally released and resulted in one of the largest known human anthrax outbreaks. In order to understand this outbreak and others, we generated a Bacillus anthracis population genetic database based upon whole-genome analysis to identify all single-nucleotide polymorphisms (SNPs) across a reference genome. Phylogenetic analysis has defined three major clades (A, B, and C), B and C being relatively rare compared to A. The A clade has numerous subclades, including a major polytomy named the trans-Eurasian (TEA) group. The TEA radiation is a dominant evolutionary feature of B. anthracis, with many contemporary populations having resulted from a large spatial dispersal of spores from a single source. Two autopsy specimens from the Sverdlovsk outbreak were deep sequenced to produce draft B. anthracis genomes. This allowed the phylogenetic placement of the Sverdlovsk strain into a clade with two Asian live vaccine strains, including the Russian Tsiankovskii strain. The genome was examined for evidence of drug resistance manipulation or other genetic engineering, but none was found. The Soviet Sverdlovsk strain genome is consistent with a wild-type strain from Russia that had no evidence of genetic manipulation during its industrial production. This work provides insights into the world’s largest biological weapons program and provides an extensive B. anthracis phylogenetic reference. PMID:27677796

  11. A Bacillus anthracis Genome Sequence from the Sverdlovsk 1979 Autopsy Specimens

    Directory of Open Access Journals (Sweden)

    Jason W. Sahl

    2016-09-01

    Full Text Available Anthrax is a zoonotic disease that occurs naturally in wild and domestic animals but has been used by both state-sponsored programs and terrorists as a biological weapon. A Soviet industrial production facility in Sverdlovsk, USSR, proved deficient in 1979 when a plume of spores was accidentally released and resulted in one of the largest known human anthrax outbreaks. In order to understand this outbreak and others, we generated a Bacillus anthracis population genetic database based upon whole-genome analysis to identify all single-nucleotide polymorphisms (SNPs across a reference genome. Phylogenetic analysis has defined three major clades (A, B, and C, B and C being relatively rare compared to A. The A clade has numerous subclades, including a major polytomy named the trans-Eurasian (TEA group. The TEA radiation is a dominant evolutionary feature of B. anthracis, with many contemporary populations having resulted from a large spatial dispersal of spores from a single source. Two autopsy specimens from the Sverdlovsk outbreak were deep sequenced to produce draft B. anthracis genomes. This allowed the phylogenetic placement of the Sverdlovsk strain into a clade with two Asian live vaccine strains, including the Russian Tsiankovskii strain. The genome was examined for evidence of drug resistance manipulation or other genetic engineering, but none was found. The Soviet Sverdlovsk strain genome is consistent with a wild-type strain from Russia that had no evidence of genetic manipulation during its industrial production. This work provides insights into the world’s largest biological weapons program and provides an extensive B. anthracis phylogenetic reference.

  12. Bacillus anthracis Capsular Conjugates Elicit Chimpanzee Polyclonal Antibodies That Protect Mice from Pulmonary Anthrax.

    Science.gov (United States)

    Chen, Zhaochun; Schneerson, Rachel; Lovchik, Julie A; Dai, Zhongdong; Kubler-Kielb, Joanna; Agulto, Liane; Leppla, Stephen H; Purcell, Robert H

    2015-08-01

    The immunogenicity of Bacillus anthracis capsule (poly-γ-D-glutamic acid [PGA]) conjugated to recombinant B. anthracis protective antigen (rPA) or to tetanus toxoid (TT) was evaluated in two anthrax-naive juvenile chimpanzees. In a previous study of these conjugates, highly protective monoclonal antibodies (MAbs) against PGA were generated. This study examines the polyclonal antibody response of the same animals. Preimmune antibodies to PGA with titers of >10(3) were detected in the chimpanzees. The maximal titer of anti-PGA was induced within 1 to 2 weeks following the 1st immunization, with no booster effects following the 2nd and 3rd immunizations. Thus, the anti-PGA response in the chimpanzees resembled a secondary immune response. Screening of sera from nine unimmunized chimpanzees and six humans revealed antibodies to PGA in all samples, with an average titer of 10(3). An anti-PA response was also observed following immunization with PGA-rPA conjugate, similar to that seen following immunization with rPA alone. However, in contrast to anti-PGA, preimmune anti-PA antibody titers and those following the 1st immunization were ≤300, with the antibodies peaking above 10(4) following the 2nd immunization. The polyclonal anti-PGA shared the MAb 11D epitope and, similar to the MAbs, exerted opsonophagocytic killing of B. anthracis. Most important, the PGA-TT-induced antibodies protected mice from a lethal challenge with virulent B. anthracis spores. Our data support the use of PGA conjugates, especially PGA-rPA targeting both toxin and capsule, as expanded-spectrum anthrax vaccines.

  13. Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.; Liu, Shihui [National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States); Leppla, Stephen H., E-mail: sleppla@niaid.nih.gov [National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Non-infectious and protease-deficient Bacillus anthracis protein expression system. Black-Right-Pointing-Pointer Successful expression and purification of a tumor-targeted fusion protein drug. Black-Right-Pointing-Pointer Very low endotoxin contamination of purified protein. Black-Right-Pointing-Pointer Efficient protein secretion simplifies purification. Black-Right-Pointing-Pointer Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGF{alpha}). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGF{alpha}). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

  14. Recombinant Expression and Purification of a Tumor-Targeted Toxin in Bacillus anthracis

    OpenAIRE

    Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.; Liu, Shihui; Leppla, Stephen H.

    2012-01-01

    Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple pr...

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

    Science.gov (United States)

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

    2016-01-01

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

  16. Allelic variation on murine chromosome 11 modifies host inflammatory responses and resistance to Bacillus anthracis.

    Science.gov (United States)

    Terra, Jill K; France, Bryan; Cote, Christopher K; Jenkins, Amy; Bozue, Joel A; Welkos, Susan L; Bhargava, Ragini; Ho, Chi-Lee; Mehrabian, Margarete; Pan, Calvin; Lusis, Aldons J; Davis, Richard C; LeVine, Steven M; Bradley, Kenneth A

    2011-12-01

    Anthrax is a potentially fatal disease resulting from infection with Bacillus anthracis. The outcome of infection is influenced by pathogen-encoded virulence factors such as lethal toxin (LT), as well as by genetic variation within the host. To identify host genes controlling susceptibility to anthrax, a library of congenic mice consisting of strains with homozygous chromosomal segments from the LT-responsive CAST/Ei strain introgressed on a LT-resistant C57BL/6 (B6) background was screened for response to LT. Three congenic strains containing CAST/Ei regions of chromosome 11 were identified that displayed a rapid inflammatory response to LT similar to, but more severe than that driven by a LT-responsive allele of the inflammasome constituent NRLP1B. Importantly, increased response to LT in congenic mice correlated with greater resistance to infection by the Sterne strain of B. anthracis. The genomic region controlling the inflammatory response to LT was mapped to 66.36-74.67 Mb on chromosome 11, a region that encodes the LT-responsive CAST/Ei allele of Nlrp1b. However, known downstream effects of NLRP1B activation, including macrophage pyroptosis, cytokine release, and leukocyte infiltration could not fully explain the response to LT or the resistance to B. anthracis Sterne in congenic mice. Further, the exacerbated response in congenic mice is inherited in a recessive manner while the Nlrp1b-mediated response to LT is dominant. Finally, congenic mice displayed increased responsiveness in a model of sepsis compared with B6 mice. In total, these data suggest that allelic variation of one or more chromosome 11 genes in addition to Nlrp1b controls the severity of host response to multiple inflammatory stimuli and contributes to resistance to B. anthracis Sterne. Expression quantitative trait locus analysis revealed 25 genes within this region as high priority candidates for contributing to the host response to LT.

  17. Allelic variation on murine chromosome 11 modifies host inflammatory responses and resistance to Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Jill K Terra

    2011-12-01

    Full Text Available Anthrax is a potentially fatal disease resulting from infection with Bacillus anthracis. The outcome of infection is influenced by pathogen-encoded virulence factors such as lethal toxin (LT, as well as by genetic variation within the host. To identify host genes controlling susceptibility to anthrax, a library of congenic mice consisting of strains with homozygous chromosomal segments from the LT-responsive CAST/Ei strain introgressed on a LT-resistant C57BL/6 (B6 background was screened for response to LT. Three congenic strains containing CAST/Ei regions of chromosome 11 were identified that displayed a rapid inflammatory response to LT similar to, but more severe than that driven by a LT-responsive allele of the inflammasome constituent NRLP1B. Importantly, increased response to LT in congenic mice correlated with greater resistance to infection by the Sterne strain of B. anthracis. The genomic region controlling the inflammatory response to LT was mapped to 66.36-74.67 Mb on chromosome 11, a region that encodes the LT-responsive CAST/Ei allele of Nlrp1b. However, known downstream effects of NLRP1B activation, including macrophage pyroptosis, cytokine release, and leukocyte infiltration could not fully explain the response to LT or the resistance to B. anthracis Sterne in congenic mice. Further, the exacerbated response in congenic mice is inherited in a recessive manner while the Nlrp1b-mediated response to LT is dominant. Finally, congenic mice displayed increased responsiveness in a model of sepsis compared with B6 mice. In total, these data suggest that allelic variation of one or more chromosome 11 genes in addition to Nlrp1b controls the severity of host response to multiple inflammatory stimuli and contributes to resistance to B. anthracis Sterne. Expression quantitative trait locus analysis revealed 25 genes within this region as high priority candidates for contributing to the host response to LT.

  18. Molecular modeling toward selective inhibitors of dihydrofolate reductase from the biological warfare agent Bacillus anthracis.

    Science.gov (United States)

    Giacoppo, Juliana O S; Mancini, Daiana T; Guimarães, Ana P; Gonçalves, Arlan S; da Cunha, Elaine F F; França, Tanos C C; Ramalho, Teodorico C

    2015-02-16

    In the present work, we applied docking and molecular dynamics techniques to study 11 compounds inside the enzymes dihydrofolate reductase (DHFR) from the biological warfare agent Bacillus anthracis (BaDHFR) and Homo sapiens sapiens (HssDHFR). Six of these compounds were selected for a study with the mutant BaF96IDHFR. Our results corroborated with experimental data and allowed the proposition of a new molecule with potential activity and better selectivity for BaDHFR.

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

    Science.gov (United States)

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

    2016-04-01

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

  20. Automated thermochemolysis reactor for detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Li, Dan [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States); Rands, Anthony D.; Losee, Scott C. [Torion Technologies, American Fork, UT 84003 (United States); Holt, Brian C. [Department of Statistics, Brigham Young University, Provo, UT 84602 (United States); Williams, John R. [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States); Lammert, Stephen A. [Torion Technologies, American Fork, UT 84003 (United States); Robison, Richard A. [Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602 (United States); Tolley, H. Dennis [Department of Statistics, Brigham Young University, Provo, UT 84602 (United States); Lee, Milton L., E-mail: milton_lee@byu.edu [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States)

    2013-05-02

    Graphical abstract: -- Highlights: •An automated sample preparation system for Bacillus anthracis endospores was developed. •A thermochemolysis method was applied to produce and derivatize biomarkers for Bacillus anthracis detection. •The autoreactor controlled the precise delivery of reagents, and TCM reaction times and temperatures. •Solid phase microextraction was used to extract biomarkers, and GC–MS was used for final identification. •This autoreactor was successfully applied to the identification of Bacillus anthracis endospores. -- Abstract: An automated sample preparation system was developed and tested for the rapid detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry (GC–MS) for eventual use in the field. This reactor is capable of automatically processing suspected bio-threat agents to release and derivatize unique chemical biomarkers by thermochemolysis (TCM). The system automatically controls the movement of sample vials from one position to another, crimping of septum caps onto the vials, precise delivery of reagents, and TCM reaction times and temperatures. The specific operations of introduction of sample vials, solid phase microextraction (SPME) sampling, injection into the GC–MS system, and ejection of used vials from the system were performed manually in this study, although they can be integrated into the automated system. Manual SPME sampling is performed by following visual and audible signal prompts for inserting the fiber into and retracting it from the sampling port. A rotating carousel design allows for simultaneous sample collection, reaction, biomarker extraction and analysis of sequential samples. Dipicolinic acid methyl ester (DPAME), 3-methyl-2-butenoic acid methyl ester (a fragment of anthrose) and two methylated sugars were used to compare the performance of the autoreactor with manual TCM. Statistical algorithms were used to construct reliable bacterial endospore signatures, and 24

  1. Modeling the potential distribution of Bacillus anthracis under multiple climate change scenarios for Kazakhstan.

    Directory of Open Access Journals (Sweden)

    Timothy Andrew Joyner

    Full Text Available Anthrax, caused by the bacterium Bacillus anthracis, is a zoonotic disease that persists throughout much of the world in livestock, wildlife, and secondarily infects humans. This is true across much of Central Asia, and particularly the Steppe region, including Kazakhstan. This study employed the Genetic Algorithm for Rule-set Prediction (GARP to model the current and future geographic distribution of Bacillus anthracis in Kazakhstan based on the A2 and B2 IPCC SRES climate change scenarios using a 5-variable data set at 55 km(2 and 8 km(2 and a 6-variable BioClim data set at 8 km(2. Future models suggest large areas predicted under current conditions may be reduced by 2050 with the A2 model predicting approximately 14-16% loss across the three spatial resolutions. There was greater variability in the B2 models across scenarios predicting approximately 15% loss at 55 km(2, approximately 34% loss at 8 km(2, and approximately 30% loss with the BioClim variables. Only very small areas of habitat expansion into new areas were predicted by either A2 or B2 in any models. Greater areas of habitat loss are predicted in the southern regions of Kazakhstan by A2 and B2 models, while moderate habitat loss is also predicted in the northern regions by either B2 model at 8 km(2. Anthrax disease control relies mainly on livestock vaccination and proper carcass disposal, both of which require adequate surveillance. In many situations, including that of Kazakhstan, vaccine resources are limited, and understanding the geographic distribution of the organism, in tandem with current data on livestock population dynamics, can aid in properly allocating doses. While speculative, contemplating future changes in livestock distributions and B. anthracis spore promoting environments can be useful for establishing future surveillance priorities. This study may also have broader applications to global public health surveillance relating to other diseases in addition to B

  2. Modeling the potential distribution of Bacillus anthracis under multiple climate change scenarios for Kazakhstan.

    Science.gov (United States)

    Joyner, Timothy Andrew; Lukhnova, Larissa; Pazilov, Yerlan; Temiralyeva, Gulnara; Hugh-Jones, Martin E; Aikimbayev, Alim; Blackburn, Jason K

    2010-03-09

    Anthrax, caused by the bacterium Bacillus anthracis, is a zoonotic disease that persists throughout much of the world in livestock, wildlife, and secondarily infects humans. This is true across much of Central Asia, and particularly the Steppe region, including Kazakhstan. This study employed the Genetic Algorithm for Rule-set Prediction (GARP) to model the current and future geographic distribution of Bacillus anthracis in Kazakhstan based on the A2 and B2 IPCC SRES climate change scenarios using a 5-variable data set at 55 km(2) and 8 km(2) and a 6-variable BioClim data set at 8 km(2). Future models suggest large areas predicted under current conditions may be reduced by 2050 with the A2 model predicting approximately 14-16% loss across the three spatial resolutions. There was greater variability in the B2 models across scenarios predicting approximately 15% loss at 55 km(2), approximately 34% loss at 8 km(2), and approximately 30% loss with the BioClim variables. Only very small areas of habitat expansion into new areas were predicted by either A2 or B2 in any models. Greater areas of habitat loss are predicted in the southern regions of Kazakhstan by A2 and B2 models, while moderate habitat loss is also predicted in the northern regions by either B2 model at 8 km(2). Anthrax disease control relies mainly on livestock vaccination and proper carcass disposal, both of which require adequate surveillance. In many situations, including that of Kazakhstan, vaccine resources are limited, and understanding the geographic distribution of the organism, in tandem with current data on livestock population dynamics, can aid in properly allocating doses. While speculative, contemplating future changes in livestock distributions and B. anthracis spore promoting environments can be useful for establishing future surveillance priorities. This study may also have broader applications to global public health surveillance relating to other diseases in addition to B. anthracis.

  3. Sample collection of virulent and non-virulent B. anthracis and Y. pestis for bioforensics analysis

    Energy Technology Data Exchange (ETDEWEB)

    Hong-geller, Elizabeth [Los Alamos National Laboratory; Valdez, Yolanda E [Los Alamos National Laboratory; Shou, Yulin [Los Alamos National Laboratory; Yoshida, Thomas M [Los Alamos National Laboratory; Marrone, Babetta L [Los Alamos National Laboratory; Dunbar, John [Los Alamos National Laboratory

    2009-01-01

    Validated sample collection methods are needed for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. To address this need, we evaluated the sample recovery efficiencies of two collection methods -- swabs and wipes -- for both non-virulent and virulent strains of B. anthracis and Y. pestis from four types of non-porous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using Real-time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface-dependent for virulent strains than non-virulent strains. For the two non-virulent strains, B. anthracis Sterne and Y. pestis A1122, collection efficiency was approximately 100% and 1 %, respectively, from all four surfaces. In contrast, recovery of B. anthracis Ames spores and Y. pestis C092 from vinyl and plastic was generally lower compared to collection from glass or stainless steel, suggesting that surface hydrophobicity may playa role in the strength of pathogen adhesion. The surface-dependent collection efficiencies observed with the virulent strains may arise from strain-specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. These findings contribute to validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.

  4. Cloning, purification and crystallization of Bacillus anthracis class C acid phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Felts, Richard L. [Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States); Reilly, Thomas J. [Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO 65211 (United States); Veterinary Medical Diagnostic Laboratory, University of Missouri-Columbia, Columbia, MO 65211 (United States); Calcutt, Michael J. [Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO 65211 (United States); Tanner, John J., E-mail: tannerjj@missouri.edu [Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States); Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States)

    2006-07-01

    Crystallization of a surface-localized acid phosphatase from Bacillus anthracis is reported. Flash annealing increased the high-resolution limit of usable data from 1.8 to 1.6 Å. Cloning, expression, purification and crystallization studies of a recombinant class C acid phosphatase from the Category A pathogen Bacillus anthracis are reported. Large diffraction-quality crystals were grown in the presence of HEPES and Jeffamine ED-2001 at pH 7.0. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.4, b = 90.1, c = 104.2 Å. The asymmetric unit is predicted to contain two protein molecules with a solvent content of 38%. Two native data sets were collected from the same crystal before and after flash-annealing. The first data set had a mosaicity of 1.6° and a high-resolution limit of 1.8 Å. After flash-annealing, the apparent mosaicity decreased to 0.9° and the high-resolution limit of usable data increased to 1.6 Å. This crystal form is currently being used to determine the structure of B. anthracis class C acid phosphatase with experimental phasing techniques.

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

    Science.gov (United States)

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

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

  6. Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis.

    Science.gov (United States)

    Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J; Liu, Shihui; Leppla, Stephen H

    2013-01-04

    Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

  7. Fieldable genotyping of Bacillus anthracis and Yersinia pestis based on 25-loci Multi Locus VNTR Analysis

    Directory of Open Access Journals (Sweden)

    Carattoli Alessandra

    2008-01-01

    Full Text Available Abstract Background Anthrax and plague are diseases caused by Bacillus anthracis and Yersinia pestis respectively. These bacteria are etiological agents for worldwide zoonotic diseases and are considered among the most feared potential bioterror agents. Strain differentiation is difficult for these microorganisms because of their high intraspecies genome homogeneity. Moreover, fast strain identification and comparison with known genotypes may be crucial for naturally occurring outbreaks versus bioterrorist events discrimination. Results Thirty-nine B. anthracis and ten Y. pestis strains, representative of the species genetic diversity, were genotyped by Agilent 2100 Bioanalyzer using previously described Multiple Locus VNTR Analysis assays (MLVA. Results were compared to previous data obtained by standard genotyping system (capillary electrophoresis on automatic sequencer and, when necessary, direct amplicon sequencing. A reference comparison table containing actual fragment sizes, sequencer sizes and Agilent sizes was produced. Conclusion In this report an automated DNA electrophoresis apparatus which provides a cheaper alternative compared to capillary electrophoresis approaches was applied for genotyping of B. anthracis and Y. pestis. This equipment, uses pre-cast gels and provides easy transportation, low maintenance and overall general logistic requirements and costs, is easy to set up and provides rapid analysis. This platform is a candidate for on-site MLVA genotyping of biothreat agents as well as other bacterial pathogens. It is an alternative to the more expensive and demanding capillary electrophoresis methods, and to the less expensive but more time-consuming classical gel electrophoresis approach.

  8. Laboratory studies on surface sampling of Bacillus anthracis contamination: summary, gaps and recommendations.

    Science.gov (United States)

    Piepel, G F; Amidan, B G; Hu, R

    2012-12-01

    This article summarizes previous laboratory studies to characterize the performance of methods for collecting, storing/transporting, processing and analysing samples from surfaces contaminated by Bacillus anthracis or related surrogates. The focus is on plate culture and count estimates of surface contamination for swab, wipe and vacuum samples of porous and nonporous surfaces. Summaries of the previous studies and their results were assessed to identify gaps in information needed as inputs to calculate key parameters critical to risk management in biothreat incidents. One key parameter is the number of samples needed to make characterization or clearance decisions with specified statistical confidence. Other key parameters include the ability to calculate, following contamination incidents, the (i) estimates of B. anthracis contamination, as well as the bias and uncertainties in the estimates and (ii) confidence in characterization and clearance decisions for contaminated or decontaminated buildings. Gaps in knowledge and understanding identified during the summary of the studies are discussed. Additional work is needed to quantify (i) the false-negative rates of surface-sampling methods with lower concentrations on various surfaces and (ii) the effects on performance characteristics of: aerosol vs liquid deposition of spores, using surrogates instead of B. anthracis, real-world vs laboratory conditions and storage and transportation conditions. Recommendations are given for future evaluations of data from existing studies and possible new studies.

  9. 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.

  10. Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase).

    Science.gov (United States)

    Rice, Amy J; Lei, Hao; Santarsiero, Bernard D; Lee, Hyun; Johnson, Michael E

    2016-10-01

    Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. Here, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (KD) and competition analysis with Ca-asp. Our results highlight that the primary difference between Class I and Class II DHOase is the catalytic loop. We also identify several compounds that can potentially be further optimized as potential B. anthracis inhibitors.

  11. Genetic diversity of Bacillus anthracis in Europe: genotyping methods in forensic and epidemiologic investigations.

    Science.gov (United States)

    Derzelle, Sylviane; Thierry, Simon

    2013-09-01

    Bacillus anthracis, the etiological agent of anthrax, a zoonosis relatively common throughout the world, can be used as an agent of bioterrorism. In naturally occurring outbreaks and in criminal release of this pathogen, a fast and accurate diagnosis is crucial to an effective response. Microbiological forensics and epidemiologic investigations increasingly rely on molecular markers, such as polymorphisms in DNA sequence, to obtain reliable information regarding the identification or source of a suspicious strain. Over the past decade, significant research efforts have been undertaken to develop genotyping methods with increased power to differentiate B. anthracis strains. A growing number of DNA signatures have been identified and used to survey B. anthracis diversity in nature, leading to rapid advances in our understanding of the global population of this pathogen. This article provides an overview of the different phylogenetic subgroups distributed across the world, with a particular focus on Europe. Updated information on the anthrax situation in Europe is reported. A brief description of some of the work in progress in the work package 5.1 of the AniBioThreat project is also presented, including (1) the development of a robust typing tool based on a suspension array technology and multiplexed single nucleotide polymorphisms scoring and (2) the typing of a collection of DNA from European isolates exchanged between the partners of the project. The know-how acquired will contribute to improving the EU's ability to react rapidly when the identity and real origin of a strain need to be established.

  12. The human-bacterial pathogen protein interaction networks of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Matthew D Dyer

    Full Text Available BACKGROUND: Bacillus anthracis, Francisella tularensis, and Yersinia pestis are bacterial pathogens that can cause anthrax, lethal acute pneumonic disease, and bubonic plague, respectively, and are listed as NIAID Category A priority pathogens for possible use as biological weapons. However, the interactions between human proteins and proteins in these bacteria remain poorly characterized leading to an incomplete understanding of their pathogenesis and mechanisms of immune evasion. METHODOLOGY: In this study, we used a high-throughput yeast two-hybrid assay to identify physical interactions between human proteins and proteins from each of these three pathogens. From more than 250,000 screens performed, we identified 3,073 human-B. anthracis, 1,383 human-F. tularensis, and 4,059 human-Y. pestis protein-protein interactions including interactions involving 304 B. anthracis, 52 F. tularensis, and 330 Y. pestis proteins that are uncharacterized. Computational analysis revealed that pathogen proteins preferentially interact with human proteins that are hubs and bottlenecks in the human PPI network. In addition, we computed modules of human-pathogen PPIs that are conserved amongst the three networks. Functionally, such conserved modules reveal commonalities between how the different pathogens interact with crucial host pathways involved in inflammation and immunity. SIGNIFICANCE: These data constitute the first extensive protein interaction networks constructed for bacterial pathogens and their human hosts. This study provides novel insights into host-pathogen interactions.

  13. Specific activation of dendritic cells enhances clearance of Bacillus anthracis following infection.

    Directory of Open Access Journals (Sweden)

    Iain J T Thompson

    Full Text Available Dendritic cells are potent activators of the immune system and have a key role in linking innate and adaptive immune responses. In the current study we have used ex vivo pulsed bone marrow dendritic cells (BMDC in a novel adoptive transfer strategy to protect against challenge with Bacillus anthracis, in a murine model. Pre-pulsing murine BMDC with either recombinant Protective Antigen (PA or CpG significantly upregulated expression of the activation markers CD40, CD80, CD86 and MHC-II. Passive transfusion of mice with pulsed BMDC, concurrently with active immunisation with rPA in alum, significantly enhanced (p<0.001 PA-specific splenocyte responses seven days post-immunisation. Parallel studies using ex vivo DCs expanded from human peripheral blood and activated under the same conditions as the murine DC, demonstrated that human DCs had a PA dose-related significant increase in the markers CD40, CD80 and CCR7 and that the increases in CD40 and CD80 were maintained when the other activating components, CpG and HK B. anthracis were added to the rPA in culture. Mice vaccinated on a single occasion intra-muscularly with rPA and alum and concurrently transfused intra-dermally with pulsed BMDC, demonstrated 100% survival following lethal B. anthracis challenge and had significantly enhanced (p<0.05 bacterial clearance within 2 days, compared with mice vaccinated with rPA and alum alone.

  14. Ecological Inference

    Science.gov (United States)

    King, Gary; Rosen, Ori; Tanner, Martin A.

    2004-09-01

    This collection of essays brings together a diverse group of scholars to survey the latest strategies for solving ecological inference problems in various fields. The last half-decade has witnessed an explosion of research in ecological inference--the process of trying to infer individual behavior from aggregate data. Although uncertainties and information lost in aggregation make ecological inference one of the most problematic types of research to rely on, these inferences are required in many academic fields, as well as by legislatures and the Courts in redistricting, by business in marketing research, and by governments in policy analysis.

  15. Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the International Space Station: a challenge for rapid and easy molecular detection of virulent B. anthracis.

    Directory of Open Access Journals (Sweden)

    Sandra P van Tongeren

    Full Text Available For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order for B. anthracis to be virulent and its close relationship to Bacillus cereus and other members of the B. cereus group. This is especially the case in environments where build-up of Bacillus spores can occur and several representatives of the B. cereus group may be present, which increases the chance for false-positives. In this study we show the presence of B. anthracis-like bacteria and other members of the B. cereus group in a microbial community within the human environment of the International Space Station and their preliminary identification by using conventional culturing as well as molecular techniques including 16S rDNA sequencing, PCR and real-time PCR. Our study shows that when monitoring the microbial hygiene in a given human environment, health risk assessment is troublesome in the case of virulent B. anthracis, especially if this should be done with rapid, easy to apply and on-site molecular methods.

  16. Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the International Space Station: a challenge for rapid and easy molecular detection of virulent B. anthracis.

    Science.gov (United States)

    van Tongeren, Sandra P; Roest, Hendrik I J; Degener, John E; Harmsen, Hermie J M

    2014-01-01

    For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order for B. anthracis to be virulent and its close relationship to Bacillus cereus and other members of the B. cereus group. This is especially the case in environments where build-up of Bacillus spores can occur and several representatives of the B. cereus group may be present, which increases the chance for false-positives. In this study we show the presence of B. anthracis-like bacteria and other members of the B. cereus group in a microbial community within the human environment of the International Space Station and their preliminary identification by using conventional culturing as well as molecular techniques including 16S rDNA sequencing, PCR and real-time PCR. Our study shows that when monitoring the microbial hygiene in a given human environment, health risk assessment is troublesome in the case of virulent B. anthracis, especially if this should be done with rapid, easy to apply and on-site molecular methods.

  17. Ecological Modernization

    NARCIS (Netherlands)

    Mol, A.P.J.

    2006-01-01

    The Encyclopedia of Globalization provides a thorough understanding of the theoretical underpinnings of globalization as well as the various historical and analytical interpretations. Consisting of over 400 entries, coverage includes key cultural, ecological, economic, geographical, historical, poli

  18. Cognitive ecology.

    Science.gov (United States)

    Hutchins, Edwin

    2010-10-01

    Cognitive ecology is the study of cognitive phenomena in context. In particular, it points to the web of mutual dependence among the elements of a cognitive ecosystem. At least three fields were taking a deeply ecological approach to cognition 30 years ago: Gibson's ecological psychology, Bateson's ecology of mind, and Soviet cultural-historical activity theory. The ideas developed in those projects have now found a place in modern views of embodied, situated, distributed cognition. As cognitive theory continues to shift from units of analysis defined by inherent properties of the elements to units defined in terms of dynamic patterns of correlation across elements, the study of cognitive ecosystems will become an increasingly important part of cognitive science.

  19. Community Ecology

    CERN Document Server

    1988-01-01

    This book presents the proceedings of a workshop on community ecology organized at Davis, in April, 1986, sponsored by the Sloan Foundation. There have been several recent symposia on community ecology (Strong et. al., 1984, Diamond and Case, 1987) which have covered a wide range of topics. The goal of the workshop at Davis was more narrow: to explore the role of scale in developing a theoretical approach to understanding communities. There are a number of aspects of scale that enter into attempts to understand ecological communities. One of the most basic is organizational scale. Should community ecology proceed by building up from population biology? This question and its ramifications are stressed throughout the book and explored in the first chapter by Simon Levin. Notions of scale have long been important in understanding physical systems. Thus, in understanding the interactions of organisms with their physical environment, questions of scale become paramount. These more physical questions illustrate the...

  20. Developing an integrated proteo-genomic approach for the characterisation of biomarkers for the identification of Bacillus anthracis.

    Science.gov (United States)

    Misra, Raju V; Ahmod, Nadia Z; Parker, Robert; Fang, Min; Shah, Haroun; Gharbia, Saheer

    2012-02-01

    Bacillus anthracis is the causative agent of anthrax, an acute and often fatal disease in humans. Due to the high genomic relatedness within the Bacillus cereus group of species it is a challenge to identify B. anthracis consistently. Alternative strategies such as proteomics coupled with mass spectrometry (MS) provide a powerful approach for biomarker discovery. However, validating and evaluating these markers, particularly for genetically homogeneous species such as B. anthracis are challenging. The objective of this study is to develop a robust biomarker discovery and validation pipeline, using proteomic methodology combined with in silico and molecular approaches, to determine a biomarker list, using B. anthracis as a model. In this exploratory study we profiled the proteome of B. anthracis and genetically related species using GeLC-Liquid Chromatography MS/MS (GeLC-LC MS/MS), identifying peptides that could be used to detect B. anthracis. Peptides were filtered to remove low quality identifications. Using comparative bioinformatic approaches, matching and searching against genomic sequence data a shortlist of peptide biomarkers was determined and validated using DNA sequencing, against a panel of closely related strains, to determine marker specificity. Further validation was performed using MS quantitation methods to assess sensitivity and specificity. A biomarker discovery pipeline was successfully developed in this study, comprising four distinct stages: proteome profiling, comparative bioinformatic validation, DNA sequencing and MS validation. Using the pipeline, 5379 peptides specific for Bacillus species and 36 peptides specific for B. anthracis were identified and validated. The 36 peptides, representing 30 proteins were derived from over 15 different clusters of orthologous group categories, including proteins involved in transcription, energy production/conservation as well as multifunctional proteins. We demonstrated that the peptide biomarkers

  1. Molecular diversity of Bacillus anthracis in the Netherlands: investigating the relationship to the wordwide population using whole-genome SNP discovery

    NARCIS (Netherlands)

    Derzelle, S.; Girault, G.; Roest, H.I.J.; Koene, M.G.J.

    2015-01-01

    Bacillus anthracis, the causative agent of anthrax, has been widely described as a clonal species. Here we report the use of both canonical SNP analysis and whole-genome sequencing to characterize the phylogenetic lineages of B. anthracis from the Netherlands. Eleven strains isolated over a 25-years

  2. Draft Genome Sequence of the Nonpathogenic, Thermotolerant, and Exopolysaccharide-Producing Bacillus anthracis Strain PFAB2 from Panifala Hot Water Spring in West Bengal, India

    Science.gov (United States)

    Banerjee, Aparna; Halder, Urmi; Chaudhry, Vasvi; Varshney, Rajeev K.; Mantri, Shrikant

    2016-01-01

    Bacillus anthracis is the causative agent of fatal anthrax in both animals and humans. It is prevalently pathogenic. Here, we present a Bacillus anthracis PFAB2 strain from a relatively unexplored Panifala hot water spring in West Bengal, India. It is nonpathogenic, exopolysaccharide producing, and thermotolerant in nature. PMID:28007848

  3. A New Generation Microarray for the Simultaneous Detection and Identification of Yersinia pestis and Bacillus anthracis in Food

    Directory of Open Access Journals (Sweden)

    Noriko Goji

    2012-01-01

    Full Text Available The use of microarrays as a multiple analytic system has generated increased interest and provided a powerful analytical tool for the simultaneous detection of pathogens in a single experiment. A wide array of applications for this technology has been reported. A low density oligonucleotide microarray was generated from the genetic sequences of Y. pestis and B. anthracis and used to fabricate a microarray chip. The new generation chip, consisting of 2,240 spots in 4 quadrants with the capability of stripping/rehybridization, was designated as “Y-PESTIS/B-ANTHRACIS 4x2K Array.” The chip was tested for specificity using DNA from a panel of bacteria that may be potentially present in food. In all, 37 unique Y. pestis-specific and 83 B. anthracis-specific probes were identified. The microarray assay distinguished Y. pestis and B. anthracis from the other bacterial species tested and correctly identified the Y. pestis-specific oligonucleotide probes using DNA extracted from experimentally inoculated milk samples. Using a whole genome amplification method, the assay was able to detect as low as 1 ng genomic DNA as the start sample. The results suggest that oligonucleotide microarray can specifically detect and identify Y. pestis and B. anthracis and may be a potentially useful diagnostic tool for detecting and confirming the organisms in food during a bioterrorism event.

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

    Science.gov (United States)

    Wang, Dian-Bing; Tian, Bo; Zhang, Zhi-Ping; Deng, Jiao-Yu; Cui, Zong-Qiang; Yang, Rui-Fu; Wang, Xu-Ying; Wei, Hong-Ping; Zhang, Xian-En

    2013-04-15

    There is an urgent need for convenient, sensitive, and specific methods to detect the spores of Bacillus anthracis, the causative agent of anthrax, because of the bioterrorism threat posed by this bacterium. In this study, we firstly develop a super-paramagnetic lateral-flow immunological detection system for B. anthracis spores. This system involves the use of a portable magnetic assay reader, super-paramagnetic iron oxide particles, lateral-flow strips and two different monoclonal antibodies directed against B. anthracis spores. This detection system specifically recognises as few as 400 pure B. anthracis spores in 30 min. This system has a linear range of 4×10³-10⁶ CFU ml⁻¹ and reproducible detection limits of 200 spores mg⁻¹ milk powder and 130 spores mg⁻¹ soil for simulated samples. In addition, this approach shows no obvious cross-reaction with other related Bacillus spores, even at high concentrations, and has no significant dependence on the duration of the storage of the immunological strips. Therefore, this super-paramagnetic lateral-flow immunological detection system is a promising tool for the rapid and sensitive detection of Bacillus anthracis spores under field conditions.

  5. LytR-CpsA-Psr enzymes as determinants of Bacillus anthracis secondary cell wall polysaccharide assembly.

    Science.gov (United States)

    Liszewski Zilla, Megan; Chan, Yvonne G Y; Lunderberg, Justin Mark; Schneewind, Olaf; Missiakas, Dominique

    2015-01-01

    Bacillus anthracis, the causative agent of anthrax, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and associated proteins (BSLs) function as chain length determinants and bind to the secondary cell wall polysaccharide (SCWP). In this study, we identified the B. anthracis lcpD mutant, which displays increased chain length and S-layer assembly defects due to diminished SCWP attachment to peptidoglycan. In contrast, the B. anthracis lcpB3 variant displayed reduced cell size and chain length, which could be attributed to increased deposition of BSLs. In other bacteria, LytR-CpsA-Psr (LCP) proteins attach wall teichoic acid (WTA) and polysaccharide capsule to peptidoglycan. B. anthracis does not synthesize these polymers, yet its genome encodes six LCP homologues, which, when expressed in S. aureus, promote WTA attachment. We propose a model whereby B. anthracis LCPs promote attachment of SCWP precursors to discrete locations in the peptidoglycan, enabling BSL assembly and regulated separation of septal peptidoglycan.

  6. N-acetylglucosamine deacetylases modulate the anchoring of the gamma-glutamyl capsule to the cell wall of Bacillus anthracis.

    Science.gov (United States)

    Candela, Thomas; Balomenou, Stavroula; Aucher, Willy; Bouriotis, Vassilis; Simore, Jean-Pierre; Fouet, Agnes; Boneca, Ivo G

    2014-06-01

    Bacillus anthracis has a complex cell wall structure composed of a peptidoglycan (PG) layer to which major structures are anchored such as a neutral polysaccharide, an S-layer, and a poly-γ-D-glutamate (PDGA) capsule. Many of these structures have central roles in the biology of B. anthracis, particularly, in virulence. However, little attention has been devoted to structurally study the PG and how it is modified in the presence of these secondary cell wall components. We present here the fine structure of the PG of the encapsulated RPG1 strain harboring both pXO1 and pXO2 virulence plasmids. We show that B. anthracis has a high degree of cross-linking and its GlcNAc residues are highly modified by N-deacetylation. The PG composition is not dependent on the presence of either LPXTG proteins or the capsule. Using NMR analysis of the PG-PDGA complex, we provide evidence for the anchoring of the PDGA to the glucosamine residues. We show that anchoring of the PDGA capsule is impaired in two PG N-deacetylase mutants, Ba1961 and Ba3679. Thus, these multiple N-deactylase activities would constitute excellent drug targets in B. anthracis by simultaneously affecting its resistance to lysozyme and to phagocytosis impairing B. anthracis survival in the host.

  7. Bacillus cereus Biovar Anthracis Causing Anthrax in Sub-Saharan Africa—Chromosomal Monophyly and Broad Geographic Distribution

    Science.gov (United States)

    Mabon, Philip; Zimmermann, Fee; Lankester, Felix; Peller, Tianna; Feistner, Anna; Todd, Angelique; Herbinger, Ilka; de Nys, Hélène M.; Muyembe-Tamfun, Jean-Jacques; Karhemere, Stomy; Wittig, Roman M.; Couacy-Hymann, Emmanuel; Grunow, Roland; Calvignac-Spencer, Sébastien; Corbett, Cindi R.; Klee, Silke R.; Leendertz, Fabian H.

    2016-01-01

    Through full genome analyses of four atypical Bacillus cereus isolates, designated B. cereus biovar anthracis, we describe a distinct clade within the B. cereus group that presents with anthrax-like disease, carrying virulence plasmids similar to those of classic Bacillus anthracis. We have isolated members of this clade from different mammals (wild chimpanzees, gorillas, an elephant and goats) in West and Central Africa (Côte d’Ivoire, Cameroon, Central African Republic and Democratic Republic of Congo). The isolates shared several phenotypic features of both B. anthracis and B. cereus, but differed amongst each other in motility and their resistance or sensitivity to penicillin. They all possessed the same mutation in the regulator gene plcR, different from the one found in B. anthracis, and in addition, carry genes which enable them to produce a second capsule composed of hyaluronic acid. Our findings show the existence of a discrete clade of the B. cereus group capable of causing anthrax-like disease, found in areas of high biodiversity, which are possibly also the origin of the worldwide distributed B. anthracis. Establishing the impact of these pathogenic bacteria on threatened wildlife species will require systematic investigation. Furthermore, the consumption of wildlife found dead by the local population and presence in a domestic animal reveal potential sources of exposure to humans. PMID:27607836

  8. Ecological epigenetics.

    Science.gov (United States)

    Kilvitis, Holly J; Alvarez, Mariano; Foust, Christy M; Schrey, Aaron W; Robertson, Marta; Richards, Christina L

    2014-01-01

    Biologists have assumed that heritable variation due to DNA sequence differences (i.e., genetic variation) allows populations of organisms to be both robust and adaptable to extreme environmental conditions. Natural selection acts on the variation among different genotypes and ultimately changes the genetic composition of the population. While there is compelling evidence about the importance of genetic polymorphisms, evidence is accumulating that epigenetic mechanisms (e.g., chromatin modifications, DNA methylation) can affect ecologically important traits, even in the absence of genetic variation. In this chapter, we review this evidence and discuss the consequences of epigenetic variation in natural populations. We begin by defining the term epigenetics, providing a brief overview of various epigenetic mechanisms, and noting the potential importance of epigenetics in the study of ecology. We continue with a review of the ecological epigenetics literature to demonstrate what is currently known about the amount and distribution of epigenetic variation in natural populations. Then, we consider the various ecological contexts in which epigenetics has proven particularly insightful and discuss the potential evolutionary consequences of epigenetic variation. Finally, we conclude with suggestions for future directions of ecological epigenetics research.

  9. Genetic diversity among Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis strains using repetitive element polymorphism-PCR.

    Science.gov (United States)

    Brumlik, Michael J; Bielawska-Drózd, Agata; Zakowska, Dorota; Liang, Xudong; Spalletta, Ronald A; Patra, Guy; Delvecchio, Vito G

    2004-01-01

    Repetitive element polymorphism-PCR (REP-PCR) is one of the tools that has been used to elucidate genetic diversity of related microorganisms. Using the MB1 primer, REP-PCR fingerprints from 110 Bacillus strains within the "B. cereus group" have identified eighteen distinct categories, while other more distantly related bacterial species fell within six additional categories. All Bacillus anthracis strains tested were found to be monomorphic by fluorophore-enhanced REP-PCR (FERP) fingerprinting using the MB1 primer. In contrast, other non- B. anthracis isolates displayed a high degree of polymorphism. Dendrogramic analysis revealed that the non- B. anthracis strains possessing the Ba813 chromosomal marker were divided into two clusters. One of the clusters shared identity with the B. cereus strains examined.

  10. Surface protein IsdC and Sortase B are required for heme-iron scavenging of Bacillus anthracis.

    Science.gov (United States)

    Maresso, Anthony W; Chapa, Travis J; Schneewind, Olaf

    2006-12-01

    Bacillus anthracis, the spore-forming agent of anthrax, requires iron for growth and is capable of scavenging heme-iron during infection. We show here that the B. anthracis iron-regulated surface determinants (isd) locus encompasses isdC, specifying a heme-iron binding surface protein. Anchoring of IsdC to the cell wall envelopes of vegetative bacilli requires srtB, which encodes sortase B. Purified sortase B cleaves IsdC between the threonine and the glycine of its NPKTG motif sorting signal. B. anthracis variants lacking either isdC or srtB display defects in heme-iron scavenging, suggesting that IsdC binding to heme-iron in the cell wall envelope contributes to bacterial uptake of heme.

  11. XML A Simplified Van Erth Single Nucleotide Polymorphism (SNP Typing Method of Bacillus Anthracis Applicable by Traditional Thermocycler Machines

    Directory of Open Access Journals (Sweden)

    Najafi Olya, Z. (BSc

    2015-05-01

    Full Text Available SNP typing is now a well-established genotyping system in Bacillus anthracis studies. In the original standard method of Van Erth, SNPs at 13 loci of the B. anthracis genome were analyzed. In order to simplify and make appropriate this expensive method to low-budget laboratory settings, 13 primer pairs targeting the 13 corresponding SNPs were designed. Besides, a universal PCR protocol was developed to enable simultaneous amplification of all loci by conventional PCR machines. The efficiency of this approach was approved by applying on nine isolates of B. anthracis. We recommend using this modified procedure as an efficient alternative to Van Erth method until developing newer and affordable techniques.

  12. Gamma-phage lysin PlyG sequence-based synthetic peptides coupled with Qdot-nanocrystals are useful for developing detection methods for Bacillus anthracis by using its surrogates, B. anthracis-Sterne and B. cereus-4342

    Directory of Open Access Journals (Sweden)

    Atreya Chintamani

    2009-07-01

    Full Text Available Abstract Background Previous reports of site-directed deletion analysis on gamma (γ-phage lysin protein (PlyG have demonstrated that removal of a short amino acid sequence in the C-terminal region encompassing a 10-amino acid motif (190LKMTADFILQ199 abrogates its binding activity specific to the cell wall of Bacillus anthracis. Whether short synthetic peptides representing the10-amino acid PlyG putative binding motif flanked by surrounding N- and C-terminal residues also selectively bind to the bacterial cell wall has not been evaluated. If such peptides do demonstrate selective binding to the cell wall, they could serve as bio-probes towards developing detection technologies for B. anthracis. Furthermore, by using B. anthracis (Sterne, 34F2, an animal vaccine and B. cereus-4342, a γ-phage susceptible rare strain as surrogates of B. anthracis, development of proof-of-concepts for B. anthracis are feasible. Results Using four different methods, we evaluated six synthetic peptides representing the putative binding motif including flanking sequences (PlyG-P1 through P6 for the bacterial cell wall binding capacity. Our analysis identified PlyG-P1, PlyG-P3 and PlyG-P5 to have binding capability to both B. anthracis (Sterne, 34F2 and B. cereus-4342. The peptides however did not bind to B. cereus-11778, B. thuringiensis, and B. cereus-10876 suggesting their specificity for B. anthracis-Sterne and B. cereus-4342. PlyG-P3 in combination with fluorescent light microscopy detected even a single bacterium in plasma spiked with the bacteria. Conclusion Overall, these studies illustrate that the short 10-amino acid sequence 'LKMTADFILQ' in fact is a stand-alone bacterial cell wall-binding motif of PlyG. In principle, synthetic peptides PlyG-P1, PlyG-P3 and PlyG-P5, especially PlyG-P3 coupled with Qdot-nanocrystals are useful as high-sensitivity bio-probes in developing detection technologies for B. anthracis.

  13. Information Ecology

    DEFF Research Database (Denmark)

    Christiansen, Ellen Tove

    2006-01-01

    The paper describes a pedagogical didactical paradigm for teaching student-designers how to deal with context issues. Form/context-relationships are conceptualized as information ecologies and described as behavioral settings using a key concept developed by social psychologist R.A. Baker...... in the 1960ties, and chosen here because it integrates cultural and psychological trajectories in a theory of living settings. The pedagogical-didactical paradigm comprises three distinct information ecologies, named after their intended outcome: the problem-setting, the exploration-setting, and the fit......-setting. It is specified how context issues can be treated within each of these information ecologies. The paper concludes by discussing the outcome of applying this paradigm with respect to the student-designers’ competence as reflective practitioners....

  14. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences.

    Science.gov (United States)

    Ågren, Joakim; Hamidjaja, Raditijo A; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; Van Rotterdam, Bart; Derzelle, Sylviane

    2013-11-15

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays.

  15. Fate of Bacillus anthracis during production of laboratory-scale cream cheese and homemade-style yoghurt.

    Science.gov (United States)

    Mertens, Katja; Schneider, Oda; Schmoock, Gernot; Melzer, Falk; Elschner, Mandy C

    2015-04-01

    The viability of Bacillus anthracis during production and storage of cream cheese and yoghurt was evaluated. Experimental cheeses were manufactured from whole milk inoculated with a suspension of B. anthracis vegetative cells and spores at a final concentration of 10(4) cfu/ml. Lactic acid bacteria (LAB) and lab ferment were used to induce milk ripening and milk coagulation. The pH-value of the contaminated milk dropped below 4.5 within the first 6 h and the amount of LAB increased by approximately 2-logs. During cheese production and storage at 5-9 °C for 24 days no growth of B. anthracis was observed. The amount of vegetative cells and spores fluctuated by 1-log. Inoculation of whole milk with heat-treated spores at 10(4) cfu/ml resulted in a slight increase of vegetative cell counts during the first 6 h. This indicated that germination occurred, but replication of vegetative cells was still inhibited in the produced cheese. Incubation of cheeses at room temperature or heating after milk coagulation strongly reduced the amount of LAB but had no effect on the growth behaviour of B. anthracis. The vegetative cell and spore content remained steady at 10(4) cfu/100 mg. During yoghurt production the pH-value decreased within 5 h below 5 and growth of B. anthracis was inhibited throughout storage. A pH-value of 5 or less is likely a critical factor to control the growth of B. anthracis. However, spores remained viable in experimental cream cheeses and yoghurts and are a potential risk of infection.

  16. [Application of the multiplex PCR and PCR-RFLP method in the identification of the Bacillus anthracis].

    Science.gov (United States)

    Szymajda, Urszula; Bartoszcze, Michał

    2005-01-01

    The aim of this study was to apply the multiplex PCR and PCR-RFLP method for the identification of the B. anthracis strains and to distinguish those bacteria from other members of the Bacillus cereus group. The multiplex PCR method enables to detect the virulence factors, i.e. the toxin and the capsule in B. anthracis strains. To do that, the authors have used 5 primer pairs specific for the fragments of lef, cya, pag genes which are present in the pXO1 plasmid and encode the toxin, the cap gene, which is present in the pXO2 plasmid and encodes the capsule, and the Ba813 chromosomal sequence. Among the four B. anthracis strains examined, three contained two plasmids and the Ba813 chromosomal sequence, while the fourth one contained the pXO1 plasmid only, together and the Ba813 chromosomal sequence. Other bacterial species, belonging to the B. cereus group, were also examined: 6 strains of B. cereus, 4 strains of B. thuringiensis and one strain of B. mycoides. The presence of Ba813 chromosomal sequence has been detected in two B. cereus strains. Neither plasmids nor Ba813 chromosomal sequence have been discovered in other B. cereus, B. thuringiensis and B. mycoides strains. The results of the survey indicate that the Ba813 chromosomal sequence does not occur solely in B. anthracis strains. The PCR-RFLP method with the use of SG-749f and SG-749r primers enabled to demonstrate the presence of DNA sequence (SG-749) in B. anthracis, B. cereus, B. thuringiensis and B. mycoides strains. Restriction analysis with enzyme AluI of the SG-749 sequence, has shown the presence of two DNA fragments at the size of about 90 and 660 bp in all B. anthracis strains. The restriction profile obtained was characteristic for B. anthracis strains and it did not occur in other investigated bacterial species belonging to the B. cereus group. It was not observed even in such B. cereus strains in which the presence of Ba813 sequence was discovered and it enabled to differentiate between B

  17. [Valuation for usefulness of selected chromosomal markers for Bacillus anthracis identification. II. Valuation for markers SSH and rpoB].

    Science.gov (United States)

    Zasada, Aleksandra Anna; Jagielski, Marek

    2006-01-01

    The article presents results of valuation for B. anthracis-specificity and usefulness for its identification obtained for different chromosomal markers. In the second part of the study markers SSH241, SSH196, SSH163, SSH133 as well as a fragment of the house-keeping gene rpoB were analyzed. For the investigation MSSCP and multiplex-PCR assays were used. There were also tested different techniques of electrophoresis. The results gave an information about specificity of tested markers and their usefulness for B. anthracis identification.

  18. Bacterial Ecology

    DEFF Research Database (Denmark)

    Fenchel, Tom

    2011-01-01

    Bacterial ecology is concerned with the interactions between bacteria and their biological and nonbiological environments and with the role of bacteria in biogeochemical element cycling. Many fundamental properties of bacteria are consequences of their small size. Thus, they can efficiently exploit...

  19. Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames

    Directory of Open Access Journals (Sweden)

    Hill Ryan E

    2009-08-01

    Full Text Available Abstract Background Bacillus anthracis is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of B. anthracis (Ames alanine racemase (AlrBax, an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native AlrBax structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation. Results B. anthracis has two open reading frames encoding for putative alanine racemases. We show that only one, dal1, is able to complement a D-alanine auxotrophic strain of E. coli. Purified Dal1, which we term AlrBax, is shown to be a dimer in solution by dynamic light scattering and has a Vmax for racemization (L- to D-alanine of 101 U/mg. The crystal structure of unmodified AlrBax is reported here to 1.95 Å resolution. Despite the overall similarity of the fold to other alanine racemases, AlrBax makes use of a chloride ion to position key active site residues for catalysis, a feature not yet observed for this enzyme in other species. Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure. Conclusion The chloride ion in AlrBax is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure. Despite differences in space group and crystal form, the two AlrBax structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.

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

    Directory of Open Access Journals (Sweden)

    Michelle M Nerandzic

    Full Text Available BACKGROUND: Removing spores of Clostridium difficile and Bacillus anthracis from skin is challenging because they are resistant to commonly used antimicrobials and soap and water washing provides only modest efficacy. We hypothesized that hygiene interventions incorporating a sporicidal electrochemically generated hypochlorous acid solution (Vashe(® would reduce the burden of spores on skin. METHODS: Hands of volunteers were inoculated with non-toxigenic C. difficile spores or B. anthracis spore surrogates to assess the effectiveness of Vashe solution for reducing spores on skin. Reduction in spores was compared for Vashe hygiene interventions versus soap and water (control. To determine the effectiveness of Vashe solution for removal of C. difficile spores from the skin of patients with C. difficile infection (CDI, reductions in levels of spores on skin were compared for soap and water versus Vashe bed baths. RESULTS: Spore removal from hands was enhanced with Vashe soak (>2.5 log10 reduction versus soap and water wash or soak (~2.0 log10 reduction; P3.5 log10 spores from hands (P<0.01 compared to washing or soaking alone. Bed baths using soap and water (N =26 patients did not reduce the percentage of positive skin cultures for CDI patients (64% before versus 57% after bathing; P =0.5, whereas bathing with Vashe solution (N =21 patients significantly reduced skin contamination (54% before versus 8% after bathing; P =0.0001. Vashe was well-tolerated with no evidence of adverse effects on skin. CONCLUSIONS: Vashe was safe and effective for reducing the burden of B. anthracis surrogates and C. difficile spores on hands. Bed baths with Vashe were effective for reducing C. difficile on skin. These findings suggest a novel strategy to reduce the burden of spores on skin.

  1. Sensing and inactivation of Bacillus anthracis Sterne by polymer-bromine complexes.

    Science.gov (United States)

    D'Angelo, Paola A; Bromberg, Lev; Hatton, T Alan; Wilusz, Eugene

    2016-08-01

    We report on the performance of brominated poly(N-vinylpyrrolidone) (PVP-Br), brominated poly(ethylene glycol) (PEG-Br), and brominated poly(allylamine-co-4-aminopyridine) (PAAm-APy-Br) for their ability to decontaminate Bacillus anthracis Sterne spores in solution while also allowing for the sensing of the spores. The polymers were brominated by bromine using carbon tetrachloride or potassium tribromide as solvents, with bromine loadings ranging from 1.6 to 4.2 mEq/g of polymer. B. anthracis Sterne spores were exposed to increasing concentrations of brominated polymers for 5 min, while the kinetics of the sporicidal activity was assessed. All brominated polymers demonstrated spore log-kills of 8 within 5 min of exposure at 12 mg/mL aqueous polymer concentration. Sensing of spores was accomplished by measuring the release of dipicolinic acid (DPA) from the spore using time-resolved fluorescence. Parent, non-brominated polymers did not cause any release of DPA and the spores remained viable. In contrast, spores exposed to the brominated polymers were inactivated and the release of DPA was observed within minutes of exposure. Also, this release of DPA continued for a long time after spore inactivation as in a controlled release process. The DPA release was more pronounced for spores exposed to brominated PVP and brominated PEG-8000 compared to brominated PAAm-APy and brominated PEG-400. Using time-resolved fluorescence, we detected as low as 2500 B. anthracis spores, with PEG-8000 being more sensitive to low spore numbers. Our results suggest that the brominated polymers may be used effectively as decontamination agents against bacterial spores while also providing the sensing capability.

  2. 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.

  3. Metal binding spectrum and model structure of the Bacillus anthracis virulence determinant MntA.

    Science.gov (United States)

    Vigonsky, Elena; Fish, Inbar; Livnat-Levanon, Nurit; Ovcharenko, Elena; Ben-Tal, Nir; Lewinson, Oded

    2015-10-01

    The potentially lethal human pathogen Bacillus anthracis expresses a putative metal import system, MntBCA, which belongs to the large family of ABC transporters. MntBCA is essential for virulence of Bacillus anthracis: deletion of MntA, the system's substrate binding protein, yields a completely non-virulent strain. Here we determined the metal binding spectrum of MntA. In contrast to what can be inferred from growth complementation studies we find no evidence that MntA binds Fe(2+) or Fe(3+). Rather, MntA binds a variety of other metal ions, including Mn(2+), Zn(2+), Cd(2+), Co(2+), and Ni(2+) with affinities ranging from 10(-6) to 10(-8) M. Binding of Zn(2+) and Co(2+) have a pronounced thermo-stabilizing effect on MntA, with Mn(2+) having a milder effect. The thermodynamic stability of MntA, competition experiments, and metal binding and release experiments all suggest that Mn(2+) is the metal that is likely transported by MntBCA and is therefore the limiting factor for virulence of Bacillus anthracis. A homology-model of MntA shows a single, highly conserved metal binding site, with four residues that participate in metal coordination: two histidines, a glutamate, and an aspartate. The metals bind to this site in a mutually exclusive manner, yet surprisingly, mutational analysis shows that for proper coordination each metal requires a different subset of these four residues. ConSurf evolutionary analysis and structural comparison of MntA and its homologues suggest that substrate binding proteins (SBPs) of metal ions use a pair of highly conserved prolines to interact with their cognate ABC transporters. This proline pair is found exclusively in ABC import systems of metal ions.

  4. Imaging B. anthracis heme catabolism in mice using the IFP1.4 gene reporter

    Science.gov (United States)

    Zhu, Banghe; Robinson, Holly; Wilganowski, Nathaniel; Nobles, Christopher L.; Sevick-Muraca, Eva; Maresso, Anthony

    2012-03-01

    B. anthracis is a gram-positive, spore-forming bacterium which likes all pathogenic bacteria, survive by sequestering heme from its host. To image B. anthracis heme catabolism in vivo, we stably transfect new red excitable fluorescent protein, IFP1.4, that requires the heme catabolism product biliverdin (BV). IFP1.4 reporter has favorable excitation and emission characteristics, which has an absorption peak at 685 nm and an emission peak at 708 nm. Therefore, IFP1.4 reporter can be imaged deeply into the tissue with less contamination from tissue autofluorescence. However, the excitation light "leakage" through optical filters can limit detection and sensitivity of IFP1.4 reporter due to the small Stoke's shift of IFP1.4 fluorescence. To minimize the excitation light leakage, an intensified CCD (ICCD) based infrared fluorescence imaging device was optimized using two band pass filters separated by a focus lens to increase the optical density at the excitation wavelength. In this study, a mouse model (DBA/J2) was first injected with B. anthracis bacteria expressing IFP1.4, 150 μl s.c., on the ventral side of the left thigh. Then mouse was given 250 μl of a 1mM BV solution via I.V. injection. Imaging was conducted as a function of time after infection under light euthanasia, excised tissues were imaged and IFP1.4 fluorescence correlated with standard culture measurements of colony forming units (CFU). The work demonstrates the use of IFP1.4 as a reporter of bacterial utilization of host heme and may provide an important tool for understanding the pathogenesis of bacterial infection and developing new anti-bacterial therapeutics.

  5. Purification, crystallization and preliminary structural analysis of nucleoside diphosphate kinase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Misra, Gauri [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India); Aggarwal, Anita [Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007 (India); Mittal, Sonia [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India); Singh, Yogendra [Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007 (India); Ramachandran, Ravishankar, E-mail: r-ravishankar@cdri.res.in [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India)

    2007-12-01

    Nucleoside diphosphate kinase from B. anthracis has been crystallized. Preliminary crystallographic analysis shows that there is one monomer in the asymmetric unit of the crystal. Bacillus anthracis nucleoside diphosphate kinase (BaNdk) is an enzyme whose primary function is to maintain deoxynucleotide triphosphate (dNTP) pools by converting deoxynucleotide diphosphates to triphosphates using ATP as the major phosphate donor. Although the structures of Ndks from a variety of organisms have been elucidated, the enzyme from sporulating bacteria has not been structurally characterized to date. Crystals of the B. anthracis enzyme were grown using the vapour-diffusion method from a hanging drop consisting of 2 µl 10 mg ml{sup −1} protein in 50 mM Tris–HCl pH 8.0, 50 mM NaCl, 5 mM EDTA equilibrated against 500 µl reservoir solution consisting of 2.25 M ammonium formate and 0.1 M HEPES buffer pH 7.25. Diffraction data extending to 2.0 Å were collected at room temperature from a single crystal with unit-cell parameters a = b = 107.53, c = 52.3 Å. The crystals are hexagonal in shape and belong to space group P6{sub 3}22. The crystals contain a monomer in the asymmetric unit, which corresponds to a Matthews coefficient (V{sub M}) of 2.1 Å{sup 3} Da{sup −1} and a solvent content of about 36.9%.

  6. 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.

  7. CXCL10 Acts as a Bifunctional Antimicrobial Molecule against Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Katie R. Margulieux

    2016-05-01

    Full Text Available Bacillus anthracis is killed by the interferon-inducible, ELR(− CXC chemokine CXCL10. Previous studies showed that disruption of the gene encoding FtsX, a conserved membrane component of the ATP-binding cassette transporter-like complex FtsE/X, resulted in resistance to CXCL10. FtsX exhibits some sequence similarity to the mammalian CXCL10 receptor, CXCR3, suggesting that the CXCL10 N-terminal region that interacts with CXCR3 may also interact with FtsX. A C-terminal truncated CXCL10 was tested to determine if the FtsX-dependent antimicrobial activity is associated with the CXCR3-interacting N terminus. The truncated CXCL10 exhibited antimicrobial activity against the B. anthracis parent strain but not the ΔftsX mutant, which supports a key role for the CXCL10 N terminus. Mutations in FtsE, the conserved ATP-binding protein of the FtsE/X complex, resulted in resistance to both CXCL10 and truncated CXCL10, indicating that both FtsX and FtsE are important. Higher concentrations of CXCL10 overcame the resistance of the ΔftsX mutant to CXCL10, suggesting an FtsX-independent killing mechanism, likely involving its C-terminal α-helix, which resembles a cationic antimicrobial peptide. Membrane depolarization studies revealed that CXCL10 disrupted membranes of the B. anthracis parent strain and the ΔftsX mutant, but only the parent strain underwent depolarization with truncated CXCL10. These findings suggest that CXCL10 is a bifunctional molecule that kills B. anthracis by two mechanisms. FtsE/X-dependent killing is mediated through an N-terminal portion of CXCL10 and is not reliant upon the C-terminal α-helix. The FtsE/X-independent mechanism involves membrane depolarization by CXCL10, likely because of its α-helix. These findings present a new paradigm for understanding mechanisms by which CXCL10 and related chemokines kill bacteria.

  8. Biochip for the Detection of Bacillus anthracis Lethal Factor and Therapeutic Agents against Anthrax Toxins

    Science.gov (United States)

    Silin, Vitalii; Kasianowicz, John J.; Michelman-Ribeiro, Ariel; Panchal, Rekha G.; Bavari, Sina; Robertson, Joseph W. F.

    2016-01-01

    Tethered lipid bilayer membranes (tBLMs) have been used in many applications, including biosensing and membrane protein structure studies. This report describes a biosensor for anthrax toxins that was fabricated through the self-assembly of a tBLM with B. anthracis protective antigen ion channels that are both the recognition element and electrochemical transducer. We characterize the sensor and its properties with electrochemical impedance spectroscopy and surface plasmon resonance. The sensor shows a sensitivity similar to ELISA and can also be used to rapidly screen for molecules that bind to the toxins and potentially inhibit their lethal effects. PMID:27348008

  9. Protein profiles of field isolates ofBacillus anthracis from different endemic areas of Indonesia

    Directory of Open Access Journals (Sweden)

    M Bhakti Poerwadikarta

    1998-03-01

    Full Text Available Sonicated cell-free extract proteins of 14 field isolates ofBacillus anthracis from six different endemic areas of Indonesia were analyzed by the use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE methods . The protein profiles of each field isolate tested demonstrated slightly different at the protein bands with molecular weights of 18, 37, 52, 65 and 70 kDa, and varied between the field isolates and vaccine strains. The variation could provide clues to the source of anthrax transmission whether it was originated from similar strain or not.

  10. Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly

    OpenAIRE

    Nguyen-Mau, Sao-Mai; Oh, So-Young; Schneewind, Daphne I.; Missiakas, Dominique; Schneewind, Olaf

    2015-01-01

    Bacillus anthracis vegetative forms assemble an S-layer comprised of two S-layer proteins, Sap and EA1. A hallmark of S-layer proteins are their C-terminal crystallization domains, which assemble into a crystalline lattice once these polypeptides are deposited on the bacterial surface via association between their N-terminal S-layer homology domains and the secondary cell wall polysaccharide. Here we show that slaQ, encoding a small cytoplasmic protein conserved among pathogenic bacilli elabo...

  11. Evaluation of the Cepheid GeneXpert System for Detecting Bacillus anthracis

    Science.gov (United States)

    2005-10-25

    anthracis Ames spores limit of detection for pXO2 using four-plex car- tridges. Cycle threshold ( LIZ CT) values are re- presented by bars and endpoint...fluorescence ( LIZ EP) values are depicted using lines. Automated biological agent diagnostics M.P. Ulrich et al. 1014 ª 2006 The Society for Applied...and Nolte, F.S. (2002) Clinical evaluation of an automated nucleic acid isolation system. Clin Chem 48, 1613–1615. Germer, J.J., Lins, M.M., Jensen , M.E

  12. Ultrasensitive electrochemical immunoassay for surface array protein, a Bacillus anthracis biomarker using Au-Pd nanocrystals loaded on boron-nitride nanosheets as catalytic labels.

    Science.gov (United States)

    Sharma, Mukesh Kumar; Narayanan, J; Pardasani, Deepak; Srivastava, Divesh N; Upadhyay, Sanjay; Goel, Ajay Kumar

    2016-06-15

    Bacillus anthracis, the causative agent of anthrax, is a well known bioterrorism agent. The determination of surface array protein (Sap), a unique biomarker for B. anthracis can offer an opportunity for specific detection of B. anthracis in culture broth. In this study, we designed a new catalytic bionanolabel and fabricated a novel electrochemical immunosensor for ultrasensitive detection of B. anthracis Sap antigen. Bimetallic gold-palladium nanoparticles were in-situ grown on poly (diallyldimethylammonium chloride) functionalized boron nitride nanosheets (Au-Pd NPs@BNNSs) and conjugated with the mouse anti-B. anthracis Sap antibodies (Ab2); named Au-Pd NPs@BNNSs/Ab2. The resulting Au-Pd NPs@BNNSs/Ab2 bionanolabel demonstrated high catalytic activity towards reduction of 4-nitrophenol. The sensitivity of the electrochemical immunosensor along with redox cycling of 4-aminophenol to 4-quinoneimine was improved to a great extent. Under optimal conditions, the proposed immunosensor exhibited a wide working range from 5 pg/mL to 100 ng/mL with a minimum detection limit of 1 pg/mL B. anthracis Sap antigen. The practical applicability of the immunosensor was demonstrated by specific detection of Sap secreted by the B. anthracis in culture broth just after 1h of growth. These labels open a new direction for the ultrasensitive detection of different biological warfare agents and their markers in different matrices.

  13. Genomic analysis of three African strains of Bacillus anthracis demonstrates that they are part of the clonal expansion of an exclusively pathogenic bacterium

    Directory of Open Access Journals (Sweden)

    L. Rouli

    2014-11-01

    Full Text Available Bacillus anthracis is the causative agent of anthrax and is classified as a ‘Category A’ biological weapon. Six complete genomes of B. anthracis (A0248, Ames, Ames Ancestor, CDC684, H0491, and Sterne are currently available. In this report, we add three African strain genomes: Sen2Col2, Sen3 and Gmb1. To study the pan‐genome of B. anthracis, we used bioinformatics tools, such as Cluster of Orthologous Groups, and performed phylogenetic analysis. We found that the three African strains contained the pX01 and pX02 plasmids, the nonsense mutation in the plcR gene and the four known prophages. These strains are most similar to the CDC684 strain and belong to the A cluster. We estimated that the B. anthracis pan‐genome has 2893 core genes (99% of the genome size and 85 accessory genes. We validated the hypothesis that B. anthracis has a closed pan‐genome and found that the three African strains carry the two plasmids associated with bacterial virulence. The pan‐genome nature of B. anthracis confirms its lack of exchange (similar to Clostridium tetani and supports its exclusively pathogenic role, despite its survival in the environment. Moreover, thanks to the study of the core content single nucleotide polymorphisms, we can see that our three African strains diverged very recently from the other B. anthracis strains.

  14. Localization and structural analysis of a conserved pyruvylated epitope in Bacillus anthracis secondary cell wall polysaccharides and characterization of the galactose-deficient wall polysaccharide from avirulent B. anthracis CDC 684.

    Science.gov (United States)

    Forsberg, L Scott; Abshire, Teresa G; Friedlander, Arthur; Quinn, Conrad P; Kannenberg, Elmar L; Carlson, Russell W

    2012-08-01

    Bacillus anthracis CDC 684 is a naturally occurring, avirulent variant and close relative of the highly pathogenic B. anthracis Vollum. Bacillus anthracis CDC 684 contains both virulence plasmids, pXO1 and pXO2, yet is non-pathogenic in animal models, prompting closer scrutiny of the molecular basis of attenuation. We structurally characterized the secondary cell wall polysaccharide (SCWP) of B. anthracis CDC 684 (Ba684) using chemical and NMR spectroscopy analysis. The SCWP consists of a HexNAc trisaccharide backbone having identical structure as that of B. anthracis Pasteur, Sterne and Ames, →4)-β-d-ManpNAc-(1 → 4)-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNAc-(1→. Remarkably, although the backbone is fully polymerized, the SCWP is the devoid of all galactosyl side residues, a feature which normally comprises 50% of the glycosyl residues on the highly galactosylated SCWPs from pathogenic strains. This observation highlights the role of defective wall assembly in virulence and indicates that polymerization occurs independently of galactose side residue attachment. Of particular interest, the polymerized Ba684 backbone retains the substoichiometric pyruvate acetal, O-acetate and amino group modifications found on SCWPs from normal B. anthracis strains, and immunofluorescence analysis confirms that SCWP expression coincides with the ability to bind the surface layer homology (SLH) domain containing S-layer protein extractable antigen-1. Pyruvate was previously demonstrated as part of a conserved epitope, mediating SLH-domain protein attachment to the underlying peptidoglycan layer. We find that a single repeating unit, located at the distal (non-reducing) end of the Ba684 SCWP, is structurally modified and that this modification is present in identical manner in the SCWPs of normal B. anthracis strains. These polysaccharides terminate in the sequence: (S)-4,6-O-(1-carboxyethylidene)-β-d-ManpNAc-(1 → 4)-[3-O-acetyl]-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNH(2)-(1→.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-06

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

  16. Literatuuronderzoek naar gegevens betreffende de betekenis van een aantal verwekkers van zoonosen in verband met de vleesconsumptie XIII Bacillus anthracis

    NARCIS (Netherlands)

    Bos JM; Engel HWB; Groothuis DG; Knapen F van; Noorle Jansen LM van; Weiss JW

    1989-01-01

    Bacillus anthracis, de verwekker van anthrax (miltvuur), komt mondiaal voor. Zowel mens als dier zijn in meer of mindere mate gevoelig voor infectie met deze bacterie. Bij dieren komen ziektebeelden voor varierend van peracute sterfte tot een meer chronisch beeld met locale laesis en aangetaste

  17. Genome Sequence of Historical Bacillus anthracis Strain Tyrol 4675 Isolated from a Bovine Anthrax Case in Austria

    Science.gov (United States)

    Antwerpen, Markus; Wölfel, Roman

    2017-01-01

    ABSTRACT In 1988, an outbreak of anthrax occurred among cattle in the Austrian state of Tyrol. Since then, Austria has been declared anthrax-free. Here, we report the draft genome sequence of one of these last outbreak strains, Bacillus anthracis Tyrol 4675, isolated from a diseased cow. PMID:28280006

  18. Technical Note: Simple, scalable, and sensitive protocol for retrieving Bacillus anthracis (and other live bacteria) from heroin.

    Science.gov (United States)

    Grass, Gregor; Ahrens, Bjoern; Schleenbecker, Uwe; Dobrzykowski, Linda; Wagner, Matthias; Krüger, Christian; Wölfel, Roman

    2016-02-01

    We describe a culture-based method suitable for isolating Bacillus anthracis and other live bacteria from heroin. This protocol was developed as a consequence of the bioforensic need to retrieve bacteria from batches of the drug associated with cases of injectional anthrax among heroin-consumers in Europe. This uncommon manifestation of infection with the notorious pathogen B. anthracis has resulted in 26 deaths between the years 2000 to 2013. Thus far, no life disease agent has been isolated from heroin during forensic investigations surrounding these incidences. Because of the conjectured very small number of disease-causing endospores in the contaminated drug it is likely that too few target sequences are available for molecular genetic analysis. Therefore, a direct culture-based approach was chosen here. Endospores of attenuated B. anthracis artificially spiked into heroin were successfully retrieved at 84-98% recovery rates using a wash solution consisting of 0.5% Tween 20 in water. Using this approach, 82 samples of un-cut heroin originating from the German Federal Criminal Police Office's heroin analysis program seized during the period between 2000 and 2014 were tested and found to be surprisingly poor in retrievable bacteria. Notably, while no B. anthracis was isolated from the drug batches, other bacteria were successfully cultured. The resulting methodical protocol is therefore suitable for analyzing un-cut heroin which can be anticipated to comprise the original microbiota from the drug's original source without interference from contaminations introduced by cutting.

  19. DnaJ sequences of Bacillus cereus strains isolated from outbreaks of hospital infection are highly similar to Bacillus anthracis.

    Science.gov (United States)

    Zhang, Jiwei; van Hung, Pham; Hayashi, Masahiro; Yoshida, Shigeru; Ohkusu, Kiyofumi; Ezaki, Takayuki

    2011-07-01

    Bacillus cereus is becoming an important nomosomial pathogen because of frequent isolation from blood cultures and from severe systemic infections. To differentiate highly pathogenic outbreak strain of B. cereus from other sources of the Bacillus cereus, we attempted to analyze their dnaJ sequences. Assays indicated that dnaJ sequence similarity of all of 52 blood culture isolates of B. cereus ranged from 92.8% to 100%. The distance between B. anthracis and B. cereus except six outbreak isolates ranged from 3.8% to 6.4%. The dnaJ sequences of six outbreak strains of B. cereus (GTC 02891, GTC 02896, GTC 02916, GTC 02917, GTC 03221, and GTC 03222) were closely related to those of B. anthracis (99.2%-99.5% sequence similarity). Ba813 sequences were only found in the six outbreak strains of B. cereus. The other pathogenic factors of B. anthracis were not found in these six outbreak strains, with the exception of GTC 02891 (cap-positive). The six outbreak strains formed clear β-hemolytic colonies on a sheep blood agar plate. Our findings suggest that outbreak strains of B. cereus isolated from blood cultures are likely to have the risk of causing serious infection, and dnaJ and Ba813 are important markers to identify such strains. Phylogenetic analysis of dnaJ and MLST revealed that the six outbreak strains of B. cereus are closely related to B. anthracis.

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

    Science.gov (United States)

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

  1. 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.

  2. Thermal inactivation of Bacillus anthracis Sterne in irradiated ground beef heated in a water bath or cooked on commercial grills

    Science.gov (United States)

    The thermal stability of heat-shocked and non heat-shocked spores of the virulence-attenuated Sterne strain of Bacillus anthracis was evaluated at select temperatures in irradiated, raw ground beef (25% fat) heated in a water bath or cooked using two different commercial grills. For the former, 3-g ...

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

    Science.gov (United States)

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

    2015-09-21

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

  4. Glycosylation of BclA Glycoprotein from Bacillus cereus and Bacillus anthracis Exosporium Is Domain-specific.

    Science.gov (United States)

    Maes, Emmanuel; Krzewinski, Frederic; Garenaux, Estelle; Lequette, Yannick; Coddeville, Bernadette; Trivelli, Xavier; Ronse, Annette; Faille, Christine; Guerardel, Yann

    2016-04-29

    The spores of the Bacillus cereus group (B. cereus, Bacillus anthracis, and Bacillus thuringiensis) are surrounded by a paracrystalline flexible yet resistant layer called exosporium that plays a major role in spore adhesion and virulence. The major constituent of its hairlike surface, the trimerized glycoprotein BclA, is attached to the basal layer through an N-terminal domain. It is then followed by a repetitive collagen-like neck bearing a globular head (C-terminal domain) that promotes glycoprotein trimerization. The collagen-like region of B. anthracis is known to be densely substituted by unusual O-glycans that may be used for developing species-specific diagnostics of B. anthracis spores and thus targeted therapeutic interventions. In the present study, we have explored the species and domain specificity of BclA glycosylation within the B. cereus group. First, we have established that the collagen-like regions of both B. anthracis and B. cereus are similarly substituted by short O-glycans that bear the species-specific deoxyhexose residues anthrose and the newly observed cereose, respectively. Second we have discovered that the C-terminal globular domains of BclA from both species are substituted by polysaccharide-like O-linked glycans whose structures are also species-specific. The presence of large carbohydrate polymers covering the surface of Bacillus spores may have a profound impact on the way that spores regulate their interactions with biotic and abiotic surfaces and represents potential new diagnostic targets.

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

    Science.gov (United States)

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

  6. Antimicrobial properties of CuO nanorods and multi-armed nanoparticles against B. anthracis vegetative cells and endospores

    Directory of Open Access Journals (Sweden)

    Pratibha Pandey

    2014-06-01

    Full Text Available Two different kinds of CuO nanoparticles (NPs namely CuO nanorods (PS2 and multi-armed nanoparticles (P5 were synthesized by wet and electrochemical routes, respectively. Their structure, morphology, size and compositions were characterized by SEM, EDX and XRD. The NPs demonstrated strong bactericidal potential against Bacillus anthracis cells and endospores. PS2 killed 92.17% of 4.5 × 104 CFU/mL B. anthracis cells within 1 h at a dose of 1 mg/mL. Whereas P5 showed a higher efficacy by killing 99.92% of 7 × 105 CFU/mL B. anthracis cells within 30 min at a dose of 0.5 mg/mL and 99.6% of 1.25 × 104 CFU/mL B. anthracis cells within 5 min at a dose of 2 mg/mL. More than 99% of spores were killed within 8 h with 2 mg/mL PS2 in LB media.

  7. A new approach to in silico SNP detection and some new SNPs in the Bacillus anthracis genome

    Directory of Open Access Journals (Sweden)

    Francoeur Joe

    2011-04-01

    Full Text Available Abstract Background Bacillus anthracis is one of the most monomorphic pathogens known. Identification of polymorphisms in its genome is essential for taxonomic classification, for determination of recent evolutionary changes, and for evaluation of pathogenic potency. Findings In this work three strains of the Bacillus anthracis genome are compared and previously unpublished single nucleotide polymorphisms (SNPs are revealed. Moreover, it is shown that, despite the highly monomorphic nature of Bacillus anthracis, the SNPs are (1 abundant in the genome and (2 distributed relatively uniformly across the sequence. Conclusions The findings support the proposition that SNPs, together with indels and variable number tandem repeats (VNTRs, can be used effectively not only for the differentiation of perfect strain data, but also for the comparison of moderately incomplete, noisy and, in some cases, unknown Bacillus anthracis strains. In the case when the data is of still lower quality, a new DNA sequence fingerprinting approach based on recently introduced markers, based on combinatorial-analytic concepts and called cyclic difference sets, can be used.

  8. Rapid detection and identification of Bacillus anthracis in food using pyrosequencing technology.

    Science.gov (United States)

    Amoako, Kingsley K; Janzen, Timothy W; Shields, Michael J; Hahn, Kristen R; Thomas, Matthew C; Goji, Noriko

    2013-08-01

    The development of advanced methodologies for the detection of Bacillus anthracis has been evolving rapidly since the release of the anthrax spores in the mail in 2001. Recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence based such as pyrosequencing, which has the capability to determine short DNA stretches in real-time using biotinylated PCR amplicons, has potential biodefense applications. Using markers from the virulence plasmids (pXO1 and pXO2) and chromosomal regions, we have demonstrated the power of this technology in the rapid, specific and sensitive detection of B. anthracis spores in food matrices including milk, juice, bottled water, and processed meat. The combined use of immunomagnetic separation and pyrosequencing showed positive detection when liquid foods (bottled water, milk, juice), and processed meat were experimentally inoculated with 6CFU/mL and 6CFU/g, respectively, without an enrichment step. Pyrosequencing is completed in about 60min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence. The entire assay (from sample preparation to sequencing information) can be completed in about 7.5h. A typical run on food samples yielded 67-80bp reads with 94-100% identity to the expected sequence. This sequence based approach is a novel application for the detection of anthrax spores in food with potential application in foodborne bioterrorism response and biodefense involving the use of anthrax spores.

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

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    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. Cloning, purification and crystallization of Bacillus anthracis class C acid phosphatase

    Science.gov (United States)

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2006-01-01

    Cloning, expression, purification and crystallization studies of a recombinant class C acid phosphatase from the Category A pathogen Bacillus anthracis are reported. Large diffraction-quality crystals were grown in the presence of HEPES and Jeffamine ED-2001 at pH 7.0. The crystals belong to space group P212121, with unit-cell parameters a = 53.4, b = 90.1, c = 104.2 Å. The asymmetric unit is predicted to contain two protein molecules with a solvent content of 38%. Two native data sets were collected from the same crystal before and after flash-annealing. The first data set had a mosaicity of 1.6° and a high-resolution limit of 1.8 Å. After flash-annealing, the apparent mosaicity decreased to 0.9° and the high-resolution limit of usable data increased to 1.6 Å. This crystal form is currently being used to determine the structure of B. anthracis class C acid phosphatase with experimental phasing techniques. PMID:16820700

  11. Laboratory studies on surface sampling of Bacillus anthracis contamination: summary, gaps, and recommendations

    Energy Technology Data Exchange (ETDEWEB)

    Piepel, Gregory F.; Amidan, Brett G.; Hu, Rebecca

    2012-12-01

    This article summarizes previous laboratory studies to characterize the performance of methods for collecting, storing/transporting, processing, and analyzing samples from surfaces contaminated by Bacillus anthracis or related surrogates. The focus is on plate culture and count estimates of surface contamination for swab, wipe, and vacuum samples of porous and nonporous surfaces. Summaries of the previous studies and their results were assessed to identify gaps in information needed as inputs to calculate key parameters critical to risk management in biothreat incidents. One key parameter is the number of samples needed to make characterization or clearance decisions with specified statistical confidence. Other key parameters include the ability to calculate, following contamination incidents, the 1) estimates of Bacillus anthracis contamination, as well as the bias and uncertainties in the estimates, and 2) confidence in characterization and clearance decisions for contaminated or decontaminated buildings. Gaps in knowledge and understanding identified during the summary of the studies are discussed. Recommendations are given for future evaluations of data from existing studies and possible new studies.

  12. Two Putative Polysaccharide Deacetylases Are Required for Osmotic Stability and Cell Shape Maintenance in Bacillus anthracis.

    Science.gov (United States)

    Arnaouteli, Sofia; Giastas, Petros; Andreou, Athina; Tzanodaskalaki, Mary; Aldridge, Christine; Tzartos, Socrates J; Vollmer, Waldemar; Eliopoulos, Elias; Bouriotis, Vassilis

    2015-05-22

    Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) β-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis.

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

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    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.

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

    Science.gov (United States)

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

    2007-04-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  16. The role of DNA restriction-modification systems in the biology of Bacillus anthracis

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    Ramakrishnan eSitaraman

    2016-01-01

    Full Text Available Restriction-modification (R-M systems are widespread among prokaryotes and, depending on their type, may be viewed as selfish genetic elements that persist as toxin-antitoxin modules or as cellular defense systems against phage infection. Studies in the last decade have made it amply clear that these two options do not exhaust the list of possible biological roles for R-M systems. Their presence in a cell may also have a bearing on other processes such as horizontal gene transfer and gene regulation. From genome sequencing and experimental data, we know that Bacillus anthracis encodes at least three methylation-dependent (typeIV restriction endonucleases, and an orphan DNA methyltransferase. In this article, we first present an outline of our current knowledge of R-M systems in Bacillus anthracis. Based on available DNA sequence data, and on our current understanding of the functions of similar genes in other systems, we conclude with hypotheses on the possible roles of the three restriction endonucleases and the orphan DNA methyltransferase.

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

    Science.gov (United States)

    Beecher, Douglas J

    2006-08-01

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

  18. A Study on molecular characterization of Razi Bacillus anthracis Sterne 34F2 substrain in Iran

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    Tadayon, K.

    2016-07-01

    Full Text Available Anthrax, a zoonotic disease caused by Bacillus anthracis, has affected humans since ancient times. For genomic characterization of Razi B. anthracis Sterne 34F2 substrain, single nucleotide polymorphism (SNP genotyping method developed by Van Erth, variable-number tandem-repeat (VNTR-8 analysis proposed by Keim, and multiple-locus VNTR analysis (MLVA-3 introduced by Levy were employed. In the SNPs typing system, where the nucleotide content of the genome at 13 evolutionary canonical loci was collectively analyzed, the originally South African 34F2 substrain was categorized in the A.Br.001/002 subgroup. In the VNTR-8 analysis, fragments with lengths of 314, 229, 162, 580, 532, 158, and 137 bp were identified at the following loci: vrrA, vrrB1, vrrB2, vrrC1, vrrC2, CG3, and pxO1, respectively. In addition, application of Levy's MLVA-3 genotyping method revealed that the genome of this strain carried 941, 451, and 864 bp fragments at AA03, AJ03, and AA07 loci, respectively. The present findings are undoubtedly helpful in meeting the requirements set by the World Organization for Animal Health (OIE and World Health Organization (WHO for anthrax vaccine manufacturers including Razi Institute. However, further similar studies are required to promote the current epidemiological knowledge of anthrax in Iran.

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

    Science.gov (United States)

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

    2015-11-01

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

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

    Science.gov (United States)

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

    2009-11-01

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

  1. Identification, Functional Characterization and Regulon Prediction of a Novel Two Component System Comprising BAS0540-BAS0541 of Bacillus anthracis

    Science.gov (United States)

    Gopalani, Monisha; Kandari, Divya; Bhatnagar, Rakesh

    2016-01-01

    Two component systems (TCSs) can be envisaged as complex molecular devices that help the bacteria to sense its environment and respond aptly. 41 TCSs are predicted in Bacillus anthracis, a potential bioterrorism agent, of which only four have been studied so far. Thus, the intricate signaling network contributed by TCSs remains largely unmapped in B. anthracis and needs comprehensive exploration. In this study, we functionally characterized one such system composed of BAS0540 (Response regulator) and BAS0541 (Histidine kinase). BAS0540-BAS0541, the closest homolog of CiaRH of Streptococcus in B. anthracis, forms a functional TCS with BAS0541 displaying autophosphorylation and subsequent phosphotransfer to BAS0540. BAS0540 was also found to accept phosphate from physiologically relevant small molecule phosphodonors like acetyl phosphate and carbamoyl phosphate. Results of qRT-PCR and immunoblotting demonstrated that BAS0540 exhibits a constitutive expression throughout the growth of B. anthracis. Regulon prediction for BAS0540 in B. anthracis was done in silico using the consensus DNA binding sequence of CiaR of Streptococcus. The predicted regulon of BAS0540 comprised of 23 genes, which could be classified into 8 functionally diverse categories. None of the proven virulence factors were a part of the predicted regulon, an observation contrasting with the regulon of CiaRH in Streptococci. Electrophoretic mobility shift assay was used to show direct binding of purified BAS0540 to the upstream regions of 5 putative regulon candidates- BAS0540 gene itself; a gene predicted to encode cell division protein FtsA; a self–immunity gene; a RND family transporter gene and a gene encoding stress (heat) responsive protein. A significant enhancement in the DNA binding ability of BAS0540 was observed upon phosphorylation. Overexpression of response regulator BAS0540 in B. anthracis led to a prodigious increase of ~6 folds in the cell length, thereby conferring it a filamentous

  2. An attenuated strain of Bacillus anthracis (CDC 684 has a large chromosomal inversion and altered growth kinetics

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    Ivins Bruce E

    2011-09-01

    Full Text Available Abstract Background An isolate originally labeled Bacillus megaterium CDC 684 was found to contain both pXO1 and pXO2, was non-hemolytic, sensitive to gamma-phage, and produced both the protective antigen and the poly-D-glutamic acid capsule. These phenotypes prompted Ezzell et al., (J. Clin. Microbiol. 28:223 to reclassify this isolate to Bacillus anthracis in 1990. Results We demonstrate that despite these B. anthracis features, the isolate is severely attenuated in a guinea pig model. This prompted whole genome sequencing and closure. The comparative analysis of CDC 684 to other sequenced B. anthracis isolates and further analysis reveals: a CDC 684 is a close relative of a virulent strain, Vollum A0488; b CDC 684 defines a new B. anthracis lineage (at least 51 SNPs that includes 15 other isolates; c the genome of CDC 684 contains a large chromosomal inversion that spans 3.3 Mbp; d this inversion has caused a displacement of the usual spatial orientation of the origin of replication (ori to the termination of replication (ter from 180° in wild-type B. anthracis to 120° in CDC 684 and e this isolate also has altered growth kinetics in liquid media. Conclusions We propose two alternative hypotheses explaining the attenuated phenotype of this isolate. Hypothesis 1 suggests that the skewed ori/ter relationship in CDC 684 has altered its DNA replication and/or transcriptome processes resulting in altered growth kinetics and virulence capacity. Hypothesis 2 suggests that one or more of the single nucleotide polymorphisms in CDC 684 has altered the expression of a regulatory element or other genes necessary for virulence.

  3. Overexpression of the pleiotropic regulator CodY decreases sporulation, attachment and pellicle formation in Bacillus anthracis.

    Science.gov (United States)

    Gopalani, Monisha; Dhiman, Alisha; Rahi, Amit; Bhatnagar, Rakesh

    2016-01-15

    CodY, a global transcriptional regulator, primarily functions as a nutrient and energy sensor. It is activated by metabolic effectors like BCAA and GTP. In low G + C Gram positive bacteria, it facilitates coupling of changes in the cellular metabolite pool with those required in the transcriptome of the cell. This pleiotropic regulator controls the expression of a vast number of genes as the cell transits from exponential to the stationary phase. Earlier studies have shown that CodY is required for the virulence of Bacillus anthracis. We sought to investigate the effect of its overexpression on the physiology of B. anthracis. In our study, we found that cellular CodY levels were unchanged during this phase-transition. Expression of endogenous CodY remained the same in different nutrient limiting conditions. Immunoblotting studies revealed CodY presence in the whole spore lysate of B. anthracis indicating it to be a component of the spore proteome. We could also detect CodY in the secretome of B. anthracis. Further, CodY was overexpressed in B. anthracis Sterne strain and this led to a 100-fold decrease in the sporulation titer and a 2.5-fold decrease in the in vitro attachment ability of the bacteria. We also observed a decrease in the pellicle formation by CodY overexpressed strain when compared to wildtype bacilli. The CodY overexpressed strain showed chaining phenotype during growth in liquid media and pellicle.

  4. Graphic Ecologies

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    Brook Weld Muller

    2014-12-01

    Full Text Available This essay describes strategic approaches to graphic representation associated with critical environmental engagement and that build from the idea of works of architecture as stitches in the ecological fabric of the city. It focuses on the building up of partial or fragmented graphics in order to describe inclusive, open-ended possibilities for making architecture that marry rich experience and responsive performance. An aphoristic approach to crafting drawings involves complex layering, conscious absence and the embracing of tension. A self-critical attitude toward the generation of imagery characterized by the notion of ‘loose precision’ may lead to more transformative and environmentally responsive architectures.

  5. The genome of a Bacillus isolate causing anthrax in chimpanzees combines chromosomal properties of B. cereus with B. anthracis virulence plasmids.

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    Silke R Klee

    Full Text Available Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as "B. cereus variety (var. anthracis".

  6. Characterization of Bacillus anthracis-Like Bacteria Isolated from Wild Great Apes from Côte d'Ivoire and Cameroon

    Science.gov (United States)

    Klee, Silke R.; Özel, Muhsin; Appel, Bernd; Boesch, Christophe; Ellerbrok, Heinz; Jacob, Daniela; Holland, Gudrun; Leendertz, Fabian H.; Pauli, Georg; Grunow, Roland; Nattermann, Herbert

    2006-01-01

    We present the microbiological and molecular characterization of bacteria isolated from four chimpanzees and one gorilla thought to have died of an anthrax-like disease in Côte d'Ivoire and Cameroon. These isolates differed significantly from classic Bacillus anthracis by the following criteria: motility, resistance to the gamma phage, and, for isolates from Cameroon, resistance to penicillin G. A capsule was expressed not only after induction by CO2 and bicarbonate but also under normal growth conditions. Subcultivation resulted in beta-hemolytic activity and gamma phage susceptibility in some subclones, suggesting differences in gene regulation compared to classic B. anthracis. The isolates from Côte d'Ivoire and Cameroon showed slight differences in their biochemical characteristics and MICs of different antibiotics but were identical in all molecular features and sequences analyzed. PCR and Southern blot analyses confirmed the presence of both the toxin and the capsule plasmid, with sizes corresponding to the B. anthracis virulence plasmids pXO1 and pXO2. Protective antigen was expressed and secreted into the culture supernatant. The isolates possessed variants of the Ba813 marker and the SG-749 fragment differing from that of classic B. anthracis strains. Multilocus sequence typing revealed a close relationship of our atypical isolates with both classic B. anthracis strains and two uncommonly virulent Bacillus cereus and Bacillus thuringiensis isolates. We propose that the newly discovered atypical B. anthracis strains share a common ancestor with classic B. anthracis or that they emerged recently by transfer of the B. anthracis plasmids to a strain of the B. cereus group. PMID:16855222

  7. Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins

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    Whittington Jessica

    2007-07-01

    Full Text Available Abstract Background The secretion time course of Bacillus anthracis strain RA3R (pXO1+/pXO2- during early, mid, and late log phase were investigated under conditions that simulate those encountered in the host. All of the identified proteins were analyzed by different software algorithms to characterize their predicted mode of secretion and cellular localization. In addition, immunogenic proteins were identified using sera from humans with cutaneous anthrax. Results A total of 275 extracellular proteins were identified by a combination of LC MS/MS and MALDI-TOF MS. All of the identified proteins were analyzed by SignalP, SecretomeP, PSORT, LipoP, TMHMM, and PROSITE to characterize their predicted mode of secretion, cellular localization, and protein domains. Fifty-three proteins were predicted by SignalP to harbor the cleavable N-terminal signal peptides and were therefore secreted via the classical Sec pathway. Twenty-three proteins were predicted by SecretomeP for secretion by the alternative Sec pathway characterized by the lack of typical export signal. In contrast to SignalP and SecretomeP predictions, PSORT predicted 171 extracellular proteins, 7 cell wall-associated proteins, and 6 cytoplasmic proteins. Moreover, 51 proteins were predicted by LipoP to contain putative Sec signal peptides (38 have SpI sites, lipoprotein signal peptides (13 have SpII sites, and N-terminal membrane helices (9 have transmembrane helices. The TMHMM algorithm predicted 25 membrane-associated proteins with one to ten transmembrane helices. Immunogenic proteins were also identified using sera from patients who have recovered from anthrax. The charge variants (83 and 63 kDa of protective antigen (PA were the most immunodominant secreted antigens, followed by charge variants of enolase and transketolase. Conclusion This is the first description of the time course of protein secretion for the pathogen Bacillus anthracis. Time course studies of protein secretion and

  8. Transcriptomic and phenotypic analysis of paralogous spx gene function in Bacillus anthracis Sterne.

    Science.gov (United States)

    Barendt, Skye; Lee, Hyunwoo; Birch, Cierra; Nakano, Michiko M; Jones, Marcus; Zuber, Peter

    2013-08-01

    Spx of Bacillus subtilis is a redox-sensitive protein, which, under disulfide stress, interacts with RNA polymerase to activate genes required for maintaining thiol homeostasis. Spx orthologs are highly conserved among low %GC Gram-positive bacteria, and often exist in multiple paralogous forms. In this study, we used B. anthracis Sterne, which harbors two paralogous spx genes, spxA1 and spxA2, to examine the phenotypes of spx null mutations and to identify the genes regulated by each Spx paralog. Cells devoid of spxA1 were sensitive to diamide and hydrogen peroxide, while the spxA1 spoxA2 double mutant was hypersensitive to the thiol-specific oxidant, diamide. Bacillus anthracis Sterne strains expressing spxA1DD or spxA2DD alleles encoding protease-resistant products were used in microarray and quantitative real-time polymerase chain reaction (RT-qPCR) analyses in order to uncover genes under SpxA1, SpxA2, or SpxA1/SpxA2 control. Comparison of transcriptomes identified many genes that were upregulated when either SpxA1DD or SpxA2DD was produced, but several genes were uncovered whose transcript levels increased in only one of the two SpxADD-expression strains, suggesting that each Spx paralog governs a unique regulon. Among genes that were upregulated were those encoding orthologs of proteins that are specifically involved in maintaining intracellular thiol homeostasis or alleviating oxidative stress. Some of these genes have important roles in B. anthracis pathogenesis, and a large number of upregulated hypothetical genes have no homology outside of the B. cereus/thuringiensis group. Microarray and RT-qPCR analyses also unveiled a regulatory link that exists between the two spx paralogous genes. The data indicate that spxA1 and spxA2 are transcriptional regulators involved in relieving disulfide stress but also control a set of genes whose products function in other cellular processes.

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

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    Simon A Weller

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

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

    Science.gov (United States)

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

    2015-01-01

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

  11. Use of high-resolution melting and melting temperature-shift assays for specific detection and identification of Bacillus anthracis based on single nucleotide discrimination.

    Science.gov (United States)

    Derzelle, Sylviane; Mendy, Christiane; Laroche, Séverine; Madani, Nora

    2011-11-01

    Single nucleotide polymorphisms (SNPs) are important diagnostic markers for the detection and differentiation of Bacillus anthracis. High-Resolution Melting (HRM) and Melting Temperature (Tm)-shift methods are two approaches that enable SNP detection without the need for expensive labeled probes. We evaluated the potential diagnostic capability of those methods to discriminate B. anthracis from the other members of the B. cereus group. Two assays targeting B. anthracis-specific SNPs in the plcR and gyrA genes were designed for each method and used to genotype a panel of 155 Bacilli strains. All B. anthracis isolates (n=65) were correctly and unambiguously identified. Assays also proved to be appropriate for the direct genotyping of biological samples. They could reliably detect B. anthracis in contaminated organs containing as little as 10(3)CFU/ml, corresponding to a few genome equivalents per reaction. The HRM and Tm-shift applications described here represent valuable tools for specific identification of B. anthracis at reduced cost.

  12. Ecological macroeconomics

    DEFF Research Database (Denmark)

    Røpke, Inge

    2013-01-01

    of this discussion includes contributions from actors involved with efforts to advance a ‘Green New Deal’ that repeats the idea that the promotion of green innovation and government funding of greener infrastructure would improve competitiveness and increase employment. This time ‘Green Keynesianism’ is accompanied...... to shift resources from consumption to investment and describing the role of consumer-citizens in such a change. The chapter starts by identifying the problems and challenges for an ecological macroeconomics. The next section outlines some of the shortcomings of traditional macroeconomics...... to energy, transport, housing, food and so forth. The fourth section outlines briefly the roles of consumer-citizens in relation to such sustainability transformations and the final section considers the possibilities for initiating the envisioned changes....

  13. 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.

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

    Science.gov (United States)

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

    2006-05-01

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

  15. Phages Preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: Past, Present and Future

    Directory of Open Access Journals (Sweden)

    Annika Gillis

    2014-07-01

    Full Text Available Many bacteriophages (phages have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here.

  16. In Silico Genomic Fingerprints of the Bacillus anthracis Group Obtained by Virtual Hybridization

    Science.gov (United States)

    Jaimes-Díaz, Hueman; Larios-Serrato, Violeta; Lloret-Sánchez, Teresa; Olguín-Ruiz, Gabriela; Sánchez-Vallejo, Carlos; Carreño-Durán, Luis; Maldonado-Rodríguez, Rogelio; Méndez-Tenorio, Alfonso

    2015-01-01

    In this study we evaluate the capacity of Virtual Hybridization to identify between highly related bacterial strains. Eight genomic fingerprints were obtained by virtual hybridization for the Bacillus anthracis genome set, and a set of 15,264 13-nucleotide short probes designed to produce genomic fingerprints unique for each organism. The data obtained from each genomic fingerprint were used to obtain hybridization patterns simulating a DNA microarray. Two virtual hybridization methods were used: the Direct and the Extended method to identify the number of potential hybridization sites and thus determine the minimum sensitivity value to discriminate between genomes with 99.9% similarity. Genomic fingerprints were compared using both methods and phylogenomic trees were constructed to verify that the minimum detection value is 0.000017. Results obtained from the genomic fingerprints suggest that the distribution in the trees is correct, as compared to other taxonomic methods. Specific virtual hybridization sites for each of the genomes studied were also identified. PMID:27600214

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

    Science.gov (United States)

    Raber, Ellen; Burklund, Alison

    2010-10-01

    Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination alternatives for use in a contaminated drinking water supply. The parameters were as follows: (i) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus), (ii) spore concentration in suspension (10(2) and 10(6) spores/ml), (iii) chemical characteristics of the decontaminant (sodium dichloro-S-triazinetrione dihydrate [Dichlor], hydrogen peroxide, potassium peroxymonosulfate [Oxone], sodium hypochlorite, and VirkonS), (iv) decontaminant concentration (0.01% to 5%), and (v) exposure time to decontaminant (10 min to 1 h). Results from 138 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5% and Dichlor or sodium hypochlorite at a concentration of 2% were highly effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor's desirable characteristics of high oxidation potential, high level of free chlorine, and a more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting the EPA biocide standard of greater than a 6-log kill after a 10-min exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS and Oxone were less effective as decontaminants than other options evaluated in this study and did not meet the EPA's efficacy standard for a biocide, although they were found to be as effective for concentrations of 10(2) spores/ml. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult.

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

    Science.gov (United States)

    Bernhards, Casey B; Popham, David L

    2014-10-01

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

  19. [Phenotypic and genetic features of cultural-morphologic variants of Bacillus anthracis].

    Science.gov (United States)

    Tsygankova, O I; Eremenko, E I; Tsygankova, E A; Buravtseva, N P; Riazanova, A G

    2008-01-01

    Comparative analysis of MVLA-genotypes of 6 Bacillus anthracis strains and 40 their variants differing on capsule- and toxin synthesis, hemolytic, proteolytic and lecitinase activity, nutritional requirements, susceptibility to anthrax bacteriophages, virulence, immunogenicity, and presence of genes for capsule and toxin synthesis was performed. Results of phylogenetic analysis of 5 chromosome locuses and plasmid locus pXO1aat which are variable for this sample of B. anthracis cultures showed that all strains divided on 2 main clusters - A and B. Cluster A consisted of 5 genotypes whereas cluster B - of 1 genotype. All highly virulent original strains and variants with characteristic phenotype Cap(CO2)(+)(O2)(-)Tox(+)ProtA(+)Hly(+) Lec(-)Trp(+) had identical genotype in 4 groups and in 5th group differences were present only in vrrA locus. All original strains and variants with the most atypical complex of phenotypic characteristics Cap (CO2)(+)(O2)(+)Tox(-)ProtA(-)Hly(-)Lec(-)Trp(-) also had the same genotype belonging to cluster B and diverged on characteristic of 5 chromosomal VNTR locuses and pXO1aat locus from typical strains. Absence of toxin production in vitro was not related to loss of genetic determinants of toxin components. Cultures with typical characteristics, one of which was ability to produce toxin in vitro, had larger sizes of amplicons of pXO1aat locus (135 and 132 nbp), whereas atoxigenic original strains and variants with complex of atypical characteristics and identical chromosome genotype had the smallest sizes (123 bnp). All original cultures were isolated in Russia, their genotypes are described for the first time.

  20. Drug interactions with Bacillus anthracis topoisomerase IV: biochemical basis for quinolone action and resistance.

    Science.gov (United States)

    Aldred, Katie J; McPherson, Sylvia A; Wang, Pengfei; Kerns, Robert J; Graves, David E; Turnbough, Charles L; Osheroff, Neil

    2012-01-10

    Bacillus anthracis, the causative agent of anthrax, is considered a serious threat as a bioweapon. The drugs most commonly used to treat anthrax are quinolones, which act by increasing the levels of DNA cleavage mediated by topoisomerase IV and gyrase. Quinolone resistance most often is associated with specific serine mutations in these enzymes. Therefore, to determine the basis for quinolone action and resistance, we characterized wild-type B. anthracis topoisomerase IV, the GrlA(S81F) and GrlA(S81Y) quinolone-resistant mutants, and the effects of quinolones and a related quinazolinedione on these enzymes. Ser81 is believed to anchor a water-Mg(2+) bridge that coordinates quinolones to the enzyme through the C3/C4 keto acid. Consistent with this hypothesized bridge, ciprofloxacin required increased Mg(2+) concentrations to support DNA cleavage by GrlA(S81F) topoisomerase IV. The three enzymes displayed similar catalytic activities in the absence of drugs. However, the resistance mutations decreased the affinity of topoisomerase IV for ciprofloxacin and other quinolones, diminished quinolone-induced inhibition of DNA religation, and reduced the stability of the enzyme-quinolone-DNA ternary complex. Wild-type DNA cleavage levels were generated by mutant enzymes at high quinolone concentrations, suggesting that increased drug potency could overcome resistance. 8-Methyl-quinazoline-2,4-dione, which lacks the quinolone keto acid (and presumably does not require the water-Mg(2+) bridge to mediate protein interactions), was more potent than quinolones against wild-type topoisomerase IV and was equally efficacious. Moreover, it maintained high potency and efficacy against the mutant enzymes, effectively inhibited DNA religation, and formed stable ternary complexes. Our findings provide an underlying biochemical basis for the ability of quinazolinediones to overcome clinically relevant quinolone resistance mutations in bacterial type II topoisomerases.

  1. Recovery efficiency and limit of detection of aerosolized Bacillus anthracis Sterne from environmental surface samples.

    Science.gov (United States)

    Estill, Cheryl Fairfield; Baron, Paul A; Beard, Jeremy K; Hein, Misty J; Larsen, Lloyd D; Rose, Laura; Schaefer, Frank W; Noble-Wang, Judith; Hodges, Lisa; Lindquist, H D Alan; Deye, Gregory J; Arduino, Matthew J

    2009-07-01

    After the 2001 anthrax incidents, surface sampling techniques for biological agents were found to be inadequately validated, especially at low surface loadings. We aerosolized Bacillus anthracis Sterne spores within a chamber to achieve very low surface loading (ca. 3, 30, and 200 CFU per 100 cm(2)). Steel and carpet coupons seeded in the chamber were sampled with swab (103 cm(2)) or wipe or vacuum (929 cm(2)) surface sampling methods and analyzed at three laboratories. Agar settle plates (60 cm(2)) were the reference for determining recovery efficiency (RE). The minimum estimated surface concentrations to achieve a 95% response rate based on probit regression were 190, 15, and 44 CFU/100 cm(2) for sampling steel surfaces and 40, 9.2, and 28 CFU/100 cm(2) for sampling carpet surfaces with swab, wipe, and vacuum methods, respectively; however, these results should be cautiously interpreted because of high observed variability. Mean REs at the highest surface loading were 5.0%, 18%, and 3.7% on steel and 12%, 23%, and 4.7% on carpet for the swab, wipe, and vacuum methods, respectively. Precision (coefficient of variation) was poor at the lower surface concentrations but improved with increasing surface concentration. The best precision was obtained with wipe samples on carpet, achieving 38% at the highest surface concentration. The wipe sampling method detected B. anthracis at lower estimated surface concentrations and had higher RE and better precision than the other methods. These results may guide investigators to more meaningfully conduct environmental sampling, quantify contamination levels, and conduct risk assessment for humans.

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

    Science.gov (United States)

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

    2014-03-01

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

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

    Science.gov (United States)

    Bernhards, Casey B.

    2014-01-01

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

  4. 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.

  5. Wordsworthian Ecology

    Institute of Scientific and Technical Information of China (English)

    朱文宣

    2014-01-01

    Wordsworth devoted himself to the ideal of a harmonious relation between human and nature, between man and soci-ety, between man and the ego. In this sense, Wordsworth improved the development of ecology. This argument will be support-ed by the approach of eco-criticism and Heidegger’s eco-philosophy. And it is supported by the following points.The first part points out that Wordsworth’s love of nature led to his love of man, which was reflected by his care for common people. Part Two shows Wordsworth’s solicitude for dwelling. His notion of dwelling had aspect of poetic dwelling. The harmonious hu-man-nature relationship reveals thee essence of free dwelling. His poetic experiment agreed with Heidegger ’s argument on poet-ic creation. His discussion of free labour was like Heidegger’s interpretation of“merit”. Part Three tells about Wordsworth’s great effort to amend the alienated human nature by treasuring the Child’s nature, imagination and human feelings.In this way, the conclusion can be got:although it would be a huge project to reinterpret Wordsworth with the approaches of eco-criticism and Heidegger’s eco-philosophy, it is still worth making the effort.

  6. Sound Ecologies

    Directory of Open Access Journals (Sweden)

    Michelle Duffy

    2010-02-01

    Full Text Available Discussions about what constitutes ‘the rural’ invariably focus on notions of spatial location – of inhabiting spaces apart from that of the metropolitan. Deeply embedded in our images of what it means to be Australian, nonetheless our intellectual framing of ‘the rural’ as something outback and beyond has significant implications for our relations with these spaces. The relatively recent phenomenon of sea- and tree-changes has struck many unawares, and not simply because a good latté is so hard to find. Although a frivolous remark, such an apparent lack does shift our focus to a bodily scale of the rural; how is rural place re/made through our experiences of it? This article originates out of on-going research that explores the practice of listening and sound and the ways in which the body can draw attention to the intuitive, emotional, and psychoanalytical processes of subjectivity and place-making. Drawing on Nigel Thrift’s concept of an ecology of place, I suggest that contemporary heightened concerns with regards to loss and lack in rural Australia has led to a nascent emotional economy – one in which individual and intimate connections to the rural require a rethinking of how we live community and belonging. In such a terrain, what does it mean to be rural?

  7. Bacillus anthracis Diversity and Geographic Potential across Nigeria, Cameroon and Chad: Further Support of a Novel West African Lineage

    OpenAIRE

    2015-01-01

    Zoonoses, diseases affecting both humans and animals, can exert tremendous pressures on human and veterinary health systems, particularly in resource limited countries. Anthrax is one such zoonosis of concern and is a disease requiring greater public health attention in Nigeria. Here we describe the genetic diversity of Bacillus anthracis in Nigeria and compare it to Chad, Cameroon and a broader global dataset based on the multiple locus variable number tandem repeat (MLVA-25) genetic typing ...

  8. Multiple Asparagine Deamidation of Bacillus anthracis Protective Antigen Causes Charge Isoforms Whose Complexity Correlates with Reduced Biological Activity

    Science.gov (United States)

    2007-01-01

    Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour...subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1 . REPORT DATE 1 ...FEB 2007 2. REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Multiple asparagine deamidation of Bacillus anthracis protective

  9. Tertiary amides of Salinomycin: A new group of antibacterial agents against Bacillus anthracis and methicillin-resistant Staphylococcus epidermidis.

    Science.gov (United States)

    Stefańska, Joanna; Antoszczak, Michał; Stępień, Karolina; Bartoszcze, Michał; Mirski, Tomasz; Huczyński, Adam

    2015-01-01

    For the first time, a series of tertiary amides of polyether antibiotic-Salinomycin have been obtained and screened for their antibacterial activity against different strains of bacteria, including Bacillus anthracis and clinical methicillin-resistant Staphylococcus epidermidis (MRSE). Moreover, biofilm inhibition of MRSE and genotoxicity tests against Bacillus subtilis have been performed. Our studies show that Salinomycin and its some derivatives are active against tested bacteria and exhibited definitely bacteriostatic, not bactericidal activity.

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

    OpenAIRE

    2012-01-01

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

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

    Science.gov (United States)

    2013-01-01

    Phosphate Buffered Saline (PBS) Preparation .................................................................. 21 3.3 Preparation of B. anthracis Cells...of Au NPs on various food borne pathogens and reported that drugs coated with NPs were highly 4 effective against C. albicans, A. niger and A...3.2 Phosphate Buffered Saline (PBS) Preparation PBS was prepared using PBS powder packets obtained from G-biosciences (St. Louis MO). To prepare

  12. Measurement of 100 B. anthracis Ames spores within 15 minutes by SERS at the US Army Edgewood Chemical Biological Ctr.

    Science.gov (United States)

    Farquharson, Stuart; Shende, Chetan; Smith, Wayne; Huang, Hermes; Sperry, Jay; Sickler, Todd; Prugh, Amber; Guicheteau, Jason

    2014-05-01

    Since the distribution of Bacillus anthracis-Ames spores through the US Postal System, there has been a persistent fear that biological warfare agents will be used by terrorists against our military abroad and our civilians at home. While there has been substantial effort since the anthrax attack of 2001 to develop analyzers to detect this and other biological warfare agents, the analyzers remain either too slow, lack sensitivity, produce high false-positive rates, or cannot be fielded. In an effort to overcome these limitations we have been developing a surface-enhanced Raman spectroscopy system. Here we describe the use of silver nanoparticles functionalized with a short peptide to selectively capture Bacillus anthracis spores and produce SER scattering. Specifically, measurements of 100 B. anthracis-Ames spores/mL in ~25 minutes performed at the US Army's Edgewood Chemical Biological Center are presented. The measurements provide a basis for the development of systems that can detect spores collected from the air or water supplies with the potential of saving lives during a biological warfare attack.

  13. Effect of delayed anthrax vaccine dose on Bacillus anthracis protective antigen IgG response and lethal toxin neutralization activity.

    Science.gov (United States)

    Pittman, Phillip R; Fisher, Diana; Quinn, Xiaofei; Schmader, Trevor; Barrera-Oro, Julio G

    2013-10-17

    We describe the Bacillus anthracis protective antigen IgG antibody response and the B. anthracis lethal toxin neutralization activity to a delayed dose of anthrax vaccine adsorbed (AVA, BioThrax(®)) using validated assays. 373 individuals received 1, 2, or 3 priming doses, 18-24 months afterward, they received a delayed dose of AVA. Overall, 23.6% of subjects showed detectable anti-PA IgG before the boost, compared to 99.2% (P<0.0001) 28 days after the boost. Geometric mean anti-PA IgG concentration (GMC) was 1.66 μg/mL before and 887.82 μg/mL after the boost (P<0.0001). The proportion of individuals with four-fold increase in GMC following the boost ranged from 93.8% to 100%. Robust anti-PA IgG levels and B. anthracis lethal toxin neutralization activity are induced when an AVA dose is delayed as long as two years. These data support continuing with the vaccination schedule when a dose is delayed as long as two years rather than restarting the series.

  14. Quantitative Determination of Lethal Toxin Proteins in Culture Supernatant of Human Live Anthrax Vaccine Bacillus anthracis A16R.

    Science.gov (United States)

    Zai, Xiaodong; Zhang, Jun; Liu, Ju; Liu, Jie; Li, Liangliang; Yin, Ying; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-02-25

    Bacillus anthracis (B. anthracis) is the etiological agent of anthrax affecting both humans and animals. Anthrax toxin (AT) plays a major role in pathogenesis. It includes lethal toxin (LT) and edema toxin (ET), which are formed by the combination of protective antigen (PA) and lethal factor (LF) or edema factor (EF), respectively. The currently used human anthrax vaccine in China utilizes live-attenuated B. anthracis spores (A16R; pXO1+, pXO2-) that produce anthrax toxin but cannot produce the capsule. Anthrax toxins, especially LT, have key effects on both the immunogenicity and toxicity of human anthrax vaccines. Thus, determining quantities and biological activities of LT proteins expressed by the A16R strain is meaningful. Here, we explored LT expression patterns of the A16R strain in culture conditions using another vaccine strain Sterne as a control. We developed a sandwich ELISA and cytotoxicity-based method for quantitative detection of PA and LF. Expression and degradation of LT proteins were observed in culture supernatants over time. Additionally, LT proteins expressed by the A16R and Sterne strains were found to be monomeric and showed cytotoxic activity, which may be the main reason for side effects of live anthrax vaccines. Our work facilitates the characterization of anthrax vaccines components and establishment of a quality control standard for vaccine production which may ultimately help to ensure the efficacy and safety of the human anthrax vaccine A16R.

  15. The worldwide distribution of genetically and phylogenetically diverse Bacillus cereus isolates harbouring Bacillus anthracis-like plasmids.

    Science.gov (United States)

    Kaminska, Paulina Sylwia; Yernazarova, Aliya; Drewnowska, Justyna Malgorzata; Zambrowski, Grzegorz; Swiecicka, Izabela

    2015-10-01

    Bacillus cereus is a close relative of B. anthracis, the causative agent of anthrax whose pathogenic determinants are located on pXO1 and pXO2 plasmids. Bacillus anthracis-like plasmids have been also noted among B. cereus, however, genetic features of B. cereus harbouring these elements remain largely undescribed, especially from the global perspective. Herein, we present the genetic polymorphism, population structure and phylogeny of B. cereus with pXO1-/pXO2-like plasmids originating from Argentina, Kazakhstan, Kenya and Poland. The plasmids were found in about 17% of the isolates, but their frequencies and expression of replicons differed within and between populations. In the multi-locus sequence typing, the bacteria exhibited high genetic polymorphism reflected by 116 sequencing types, including 84 singletons and 10 clonal complexes, which mainly consisted of isolates of the same origin. The phylogenetic analysis of pXO1-/pXO2-like positive B. cereus isolates revealed six independent clades; in certain clades individual populations predominated. Generally, B. cereus with pXO1-/pXO2-like plasmids did not indicate the genetic relationship with B. anthracis, and cannot be classified into an evolutionary independent anthrax line within the B. cereus group. Our report is of a crucial importance for discovering the genetic specificity and evolution of B. cereus bacilli.

  16. DNA sequence conservation between the Bacillus anthracis pXO2 plasmid and genomic sequence from closely related bacteria

    Directory of Open Access Journals (Sweden)

    Sabin Robert

    2002-12-01

    Full Text Available Abstract Background Complete sequencing and annotation of the 96.2 kb Bacillus anthracis plasmid, pXO2, predicted 85 open reading frames (ORFs. Bacillus cereus and Bacillus thuringiensis isolates that ranged in genomic similarity to B. anthracis, as determined by amplified fragment length polymorphism (AFLP analysis, were examined by PCR for the presence of sequences similar to 47 pXO2 ORFs. Results The two most distantly related isolates examined, B. thuringiensis 33679 and B. thuringiensis AWO6, produced the greatest number of ORF sequences similar to pXO2; 10 detected in 33679 and 16 in AWO6. No more than two of the pXO2 ORFs were detected in any one of the remaining isolates. Dot-blot DNA hybridizations between pXO2 ORF fragments and total genomic DNA from AWO6 were consistent with the PCR assay results for this isolate and also revealed nine additional ORFs shared between these two bacteria. Sequences similar to the B. anthracis cap genes or their regulator, acpA, were not detected among any of the examined isolates. Conclusions The presence of pXO2 sequences in the other Bacillus isolates did not correlate with genomic relatedness established by AFLP analysis. The presence of pXO2 ORF sequences in other Bacillus species suggests the possibility that certain pXO2 plasmid gene functions may also be present in other closely related bacteria.

  17. Genetic Comparison of B. Anthracis and its Close Relatives Using AFLP and PCR Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, P.J.; Hill, K.K.; Laker, M.T.; Ticknor, L.O.; Keim, P.S.

    1999-02-01

    Amplified Fragment length Polymorphism (AFLP) analysis allows a rapid, relatively simple analysis of a large portion of a microbial genome, providing information about the species and its phylogenetic relationship to other microbes (Vos, et al., 1995). The method simply surveys the genome for length and sequence polymorphisms. The pattern identified can be used for comparison to the genomes of other species. Unlike other methods, it does not rely on analysis of a single genetic locus that may bias the interpretation of results and it does not require any prior knowledge of the targeted organism. Moreover, a standard set of reagents can be applied to any species without using species-specific information or molecular probes. The authors are using AFLP's to rapidly identify different bacterial species. A comparison of AFLP profiles generated from a large battery of B. anthracis strains shows very little variability among different isolates (Keim, et al., 1997). By contrast, there is a significant difference between AFLP profiles generated for any B. anthracis strain and even the most closely related Bacillus species. Sufficient variability is apparent among all known microbial species to allow phylogenetic analysis based on large numbers of genetically unlinked loci. These striking differences among AFLP profiles allow unambiguous identification of previously identified species and phylogenetic placement of newly characterized isolates relative to known species based on a large number of independent genetic loci. Data generated thus far show that the method provides phylogenetic analyses that are consistent with other widely accepted phylogenetic methods. However, AFLP analysis provides a more detailed analysis of the targets and samples a much larger portion of the genome. Consequently, it provides an inexpensive, rapid means of characterizing microbial isolates to further differentiate among strains and closely related microbial species. Such information

  18. Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

    Science.gov (United States)

    Bavykin, Sergei G.; Mirzabekov, Andrei D.

    2007-10-30

    The present invention is directed to a novel method of discriminating a highly infectious bacterium Bacillus anthracis from a group of closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations. The identification and analysis of these sequence variations enables positive discrimination of isolates of the B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed probes, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

  19. Possible Use of Bacteriophages Active against Bacillus anthracis and Other B. cereus Group Members in the Face of a Bioterrorism Threat

    Science.gov (United States)

    Weber-Dąbrowska, Beata; Borysowski, Jan; Górski, Andrzej

    2014-01-01

    Anthrax is an infectious fatal disease with epidemic potential. Nowadays, bioterrorism using Bacillus anthracis is a real possibility, and thus society needs an effective weapon to neutralize this threat. The pathogen may be easily transmitted to human populations. It is easy to store, transport, and disseminate and may survive for many decades. Recent data strongly support the effectiveness of bacteriophage in treating bacterial diseases. Moreover, it is clear that bacteriophages should be considered a potential incapacitative agent against bioterrorism using bacteria belonging to B. cereus group, especially B. anthracis. Therefore, we have reviewed the possibility of using bacteriophages active against Bacillus anthracis and other species of the B. cereus group in the face of a bioterrorism threat. PMID:25247187

  20. Possible Use of Bacteriophages Active against Bacillus anthracis and Other B. cereus Group Members in the Face of a Bioterrorism Threat

    Directory of Open Access Journals (Sweden)

    Ewa Jończyk-Matysiak

    2014-01-01

    Full Text Available Anthrax is an infectious fatal disease with epidemic potential. Nowadays, bioterrorism using Bacillus anthracis is a real possibility, and thus society needs an effective weapon to neutralize this threat. The pathogen may be easily transmitted to human populations. It is easy to store, transport, and disseminate and may survive for many decades. Recent data strongly support the effectiveness of bacteriophage in treating bacterial diseases. Moreover, it is clear that bacteriophages should be considered a potential incapacitative agent against bioterrorism using bacteria belonging to B. cereus group, especially B. anthracis. Therefore, we have reviewed the possibility of using bacteriophages active against Bacillus anthracis and other species of the B. cereus group in the face of a bioterrorism threat.

  1. Possible use of bacteriophages active against Bacillus anthracis and other B. cereus group members in the face of a bioterrorism threat.

    Science.gov (United States)

    Jończyk-Matysiak, Ewa; Kłak, Marlena; Weber-Dąbrowska, Beata; Borysowski, Jan; Górski, Andrzej

    2014-01-01

    Anthrax is an infectious fatal disease with epidemic potential. Nowadays, bioterrorism using Bacillus anthracis is a real possibility, and thus society needs an effective weapon to neutralize this threat. The pathogen may be easily transmitted to human populations. It is easy to store, transport, and disseminate and may survive for many decades. Recent data strongly support the effectiveness of bacteriophage in treating bacterial diseases. Moreover, it is clear that bacteriophages should be considered a potential incapacitative agent against bioterrorism using bacteria belonging to B. cereus group, especially B. anthracis. Therefore, we have reviewed the possibility of using bacteriophages active against Bacillus anthracis and other species of the B. cereus group in the face of a bioterrorism threat.

  2. Evaluation of PCR Systems for Field Screening of Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Ozanich, Richard M.; Colburn, Heather A.; Victry, Kristin D.; Bartholomew, Rachel A.; Arce, Jennifer S.; Heredia-Langner, Alejandro; Jarman, Kristin; Kreuzer, Helen W.; Bruckner-Lea, Cynthia J.

    2017-02-01

    There is little published data on the performance of hand-portable polymerase chain reaction (PCR) instruments that could be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated five commercially available hand-portable PCR instruments for detection of Bacillus anthracis (Ba). We designed a cost-effective, statistically-based test plan that allows instruments to be evaluated at performance levels ranging from 0.85-0.95 lower confidence bound (LCB) on the probability of detection (POD) at confidence levels of 80-95%. We assessed specificity using purified genomic DNA from 13 Ba strains and 18 Bacillus near neighbors, interference with 22 common hoax powders encountered in the field, and PCR inhibition when Ba spores were spiked into these powders. Our results indicated that three of the five instruments achieved >0.95 LCB on the POD with 95% confidence at test concentrations of 2,000 genome equivalents/mL (comparable to 2,000 spores/mL), displaying more than sufficient sensitivity for screening suspicious powders. These instruments exhibited no false positive results or PCR inhibition with common hoax powders, and reliably detected Ba spores spiked into common hoax powders, though some issues with instrument controls were observed. Our testing approach enables efficient instrument performance testing to a statistically rigorous and cost-effective test plan to generate performance data that will allow users to make informed decisions regarding the purchase and use of biodetection equipment in the field.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2008-08-01

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

  5. The Saccharomyces boulardii CNCM I-745 Strain Shows Protective Effects against the B. anthracis LT Toxin

    Directory of Open Access Journals (Sweden)

    Rodolphe Pontier-Bres

    2015-10-01

    Full Text Available The probiotic yeast Saccharomyces boulardii (S. boulardii has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax.

  6. Electrically active magnetic nanoparticles as novel concentrator and electrochemical redox transducer in Bacillus anthracis DNA detection.

    Science.gov (United States)

    Pal, Sudeshna; Alocilja, Evangelyn C

    2010-12-15

    Magnetic polymer nanostructures are a new class of multifunctional nanomaterials that are recently being explored in biosensor devices. In this paper, for the first time we report the novel application of electrically active magnetic (EAM) nanoparticles as concentrator of DNA targets as well as electrochemical transducers for detection of the Bacillus anthracis protective antigen A (pag A) gene. The EAM nanoparticles are synthesized by chemical polymerization and have dimensions of 80-100 nm. The biosensor detection encompasses two sets of DNA probes that are specific to the target gene: the detector probe labeled with the EAM nanoparticles and the biotinylated capture probe. The DNA targets are double hybridized to the detector and the capture probes and concentrated from nonspecific DNA fragments by applying a magnetic field. Subsequently, the DNA sandwiched targets (EAM-detector probe-DNA target-capture probe-biotin) are captured on streptavidin modified screen printed carbon electrodes through the biotinylated capture probes. Detection is achieved electrochemically by measuring the oxidation-reduction signal of the EAM nanoparticles. Preliminary results indicate that the biosensor is able to detect the redox signal of the EAM nanoparticles at DNA concentrations as low as 0.01 ng/μl.

  7. 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.

  8. Targeted Mutations of Bacillus anthracis Dihydrofolate Reductase Condense Complex Structure-Activity Relationships

    Energy Technology Data Exchange (ETDEWEB)

    J Beierlein; N Karri; A Anderson

    2011-12-31

    Several antifolates, including trimethoprim (TMP) and a series of propargyl-linked analogues, bind dihydrofolate reductase from Bacillus anthracis (BaDHFR) with lower affinity than is typical in other bacterial species. To guide lead optimization for BaDHFR, we explored a new approach to determine structure-activity relationships whereby the enzyme is altered and the analogues remain constant, essentially reversing the standard experimental design. Active site mutants of the enzyme, Ba(F96I)DHFR and Ba(Y102F)DHFR, were created and evaluated with enzyme inhibition assays and crystal structures. The affinities of the antifolates increase up to 60-fold with the Y102F mutant, suggesting that interactions with Tyr 102 are critical for affinity. Crystal structures of the enzymes bound to TMP and propargyl-linked inhibitors reveal the basis of TMP resistance and illuminate the influence of Tyr 102 on the lipophilic linker between the pyrimidine and aryl rings. Two new inhibitors test and validate these conclusions and show the value of the technique for providing new directions during lead optimization.

  9. Analysis of the sporicidal activity of chlorine dioxide disinfectant against Bacillus anthracis (Sterne strain)

    Science.gov (United States)

    Chatuev, B.A.; Peterson, J.W.

    2009-01-01

    Summary Routine surface decontamination is an essential hospital and laboratory procedure, but the list of effective, noncorrosive disinfectants that kill spores is limited. We investigated the sporicidal potential of an aqueous chlorine dioxide solution and encountered some unanticipated problems. Quantitative bacteriological culture methods were used to determine the log10 reduction of Bacillus anthracis (Sterne strain) spores following 3 min exposure to various concentrations of aqueous chlorine dioxide solutions at room temperature in sealed tubes, as well as spraying onto plastic and stainless steel surfaces in a biological safety cabinet. Serial 10-fold dilutions of the treated spores were then plated on 5% sheep blood agar plates, and the survivor colonies were enumerated. Disinfection of spore suspensions with aqueous chlorine dioxide solution in sealed microfuge tubes was highly effective, reducing the viable spore counts by 8 log10 in only 3 min. By contrast, the process of spraying or spreading the disinfectant onto surfaces resulted in only a 1 log10 kill because the chlorine dioxide gas was rapidly vaporised from the solutions. Full potency of the sprayed aqueous chlorine dioxide solution was restored by preparing the chlorine dioxide solution in 5% bleach (0.3% sodium hypochlorite). The volatility of chlorine dioxide can cause treatment failures that constitute a serious hazard for unsuspecting users. Supplementation of the chlorine dioxide solution with 5% bleach (0.3% sodium hypochlorite) restored full potency and increased stability for one week. PMID:20061062

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

    Science.gov (United States)

    Tufts, Jenia A McBrian; Rosati, Jacky A

    2012-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-08-01

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

  12. In Silico Genomic Fingerprints of the Bacillus anthracis Group Obtained by Virtual Hybridization

    Directory of Open Access Journals (Sweden)

    Hueman Jaimes-Díaz

    2015-02-01

    Full Text Available In this study we evaluate the capacity of Virtual Hybridization to identify between highly related bacterial strains. Eight genomic fingerprints were obtained by virtual hybridization for the Bacillus anthracis genome set, and a set of 15,264 13-nucleotide short probes designed to produce genomic fingerprints unique for each organism. The data obtained from each genomic fingerprint were used to obtain hybridization patterns simulating a DNA microarray. Two virtual hybridization methods were used: the Direct and the Extended method to identify the number of potential hybridization sites and thus determine the minimum sensitivity value to discriminate between genomes with 99.9% similarity. Genomic fingerprints were compared using both methods and phylogenomic trees were constructed to verify that the minimum detection value is 0.000017. Results obtained from the genomic fingerprints suggest that the distribution in the trees is correct, as compared to other taxonomic methods. Specific virtual hybridization sites for each of the genomes studied were also identified.

  13. First detection of Bacillus anthracis in feces of free-ranging raptors from central Argentina.

    Science.gov (United States)

    Saggese, Miguel D; Noseda, Ramón P; Uhart, Marcela M; Deem, Sharon L; Ferreyra, Hebe; Romano, Marcelo C; Ferreyra-Armas, María C; Hugh-Jones, Martin

    2007-01-01

    Prevalence of anthrax spores in feces of raptors was determined from samples collected in November-December 2000 and April-May 2001 in an agricultural region of Santa Fé province, Argentina. Feces were tested from 48 birds of six raptor species. One of 14 chimango caracaras (Milvago chimango) and one of eight road-side hawks (Buteo magnirostris) tested positive. The prevalence of Bacillus anthracis spores in feces for the six species was 4% (n=48). The prevalence was 7% (n=14) for chimango caracaras, 13% for road-side hawks (n=8), and 0% for the remaining species (Burrowing owl [Speotyto cunicularia] [n=17], Swainson's hawk [Buteo swainsoni] [n=3], Aplomado falcon [Falco femoralis] [n=2], and American kestrel [Falco sparverius] [n=4]). Grouped by their feeding habits, prevalence for scavenger species was not significantly different than for predators (7% vs. 3%, P>0.999). This study provides evidence that in central Argentina scavenger and non-scavenger raptors may have a role in the epidemiology of anthrax. Long-term studies to determine the extent of this potential involvement in the epidemiology of anthrax in central Argentina are required.

  14. Bacillus anthracis Co-Opts Nitric Oxide and Host Serum Albumin for Pathogenicity in Hypoxic Conditions

    Directory of Open Access Journals (Sweden)

    Stephen eSt John

    2013-05-01

    Full Text Available Bacillus anthracis is a dangerous pathogen of humans and many animal species. Its virulence has been mainly attributed to the production of Lethal and Edema toxins as well as the antiphagocytic capsule. Recent data indicate that the nitric oxide (NO synthase (baNOS plays an important pathogenic role at the early stage of disease by protecting bacteria from the host reactive species and S-nytrosylating the mitochondrial proteins in macrophages. In this study we for the first time present evidence that bacteria-derived NO participates in the generation of highly reactive oxidizing species which could be abolished by the NOS inhibitor L-NAME, free thiols, and superoxide dismutase but not catalase. The formation of toxicants is likely a result of the simultaneous formation of NO and superoxide leading to a labile peroxynitrite and its stable decomposition product, nitrogen dioxide. The toxicity of bacteria could be potentiated in the presence of bovine serum albumin. This effect is consistent with the property of serum albumin to serves as a trap of a volatile NO accelerating its reactions. Our data suggest that during infection in the hypoxic environment of pre-mortal host the accumulated NO is expected to have a broad toxic impact on host cell functions.

  15. Expression and Purification of the Bacillus anthracis Protective Antigen Receptor-binding Domain

    Institute of Scientific and Technical Information of China (English)

    葛猛; 徐俊杰; 李冰; 董大勇; 宋小红; 郭强; 赵剑; 陈薇

    2004-01-01

    The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E. coli. Signal sequence of the outer membrane protein A (OmpA) of E. coli was attached to the 5' end of the gene encoding protective antigen receptor-binding domain (the 4th domain of PA, PALM). The plasmid carrying the fusion gene was then transformed into E. coli and induced to express recombinant PAlM by IFFG. The recombinant protein was purified by chromatography and then identified by N-terrainal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ionexchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E. coli. The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.

  16. 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.

  17. 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.

  18. Multiplex PCR for species-level identification of Bacillus anthracis and detection of pXO1, pXO2, and related plasmids.

    Science.gov (United States)

    Riojas, Marco A; Kiss, Katalin; McKee, Marian L; Hazbón, Manzour Hernando

    2015-01-01

    The Bacillus anthracis virulence plasmids pXO1 and pXO2 have critical implications for biosafety and select agent status. The proper identification and characterization of B. anthracis and its plasmid profile is important to the biodefense research community. Multiplex PCR was used to simultaneously detect a B. anthracis-specific chromosomal mutation, 4 targets distributed across pXO1, 3 targets distributed across pXO2, and highly conserved regions of the 16S gene, allowing an internal positive control for each sample. The multiplex PCR can produce as many as 9 easily separable and distinguishable amplicons, ranging in size from 188 to 555 bp. The PCR results were used to characterize DNA samples extracted from B. anthracis, other Bacillus species, and other bacterial species from many different genera. With the exception of 2 novel putative plasmids discovered, testing against inclusion and extensive exclusion panels showed 100% correlation to previously published and expected results. Upon testing 29 previously unpublished B. anthracis strains, 10 (34.5%) were pXO1(+)/pXO2(+), 9 (31.0%) were pXO1(+)/pXO2(-), 7 (24.1%) were pXO1(-)/pXO2(+), and 3 (10.3%) were pXO1(-)/pXO2(-). The present work presents a novel 9-target multiplex PCR assay capable of species-level identification of B. anthracis via a unique chromosomal marker and the detection of pXO1 and pXO2 via multiply redundant targets on each.

  19. Development and field testing of a mobile chlorine dioxide generation system for the decontamination of buildings contaminated with Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Wood, Joseph P., E-mail: wood.joe@epa.gov [U.S. Environmental Protection Agency, Office of Research and Development, National Homeland Security Research Center, MC-E343-06, Research Triangle Park, NC 27711 (United States); Blair Martin, G., E-mail: martin.blair@epa.gov [U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, MC-E340-C, Research Triangle Park, NC 27711 (United States)

    2009-05-30

    The numerous buildings that became contaminated with Bacillus anthracis (the bacterium causing the disease anthrax) in 2001, and more recent B. anthracis - related events, point to the need to have effective decontamination technologies for buildings contaminated with biological threat agents. The U.S. Government developed a portable chlorine dioxide (ClO{sub 2}) generation system to decontaminate buildings contaminated with B. anthracis spores, and this so-called mobile decontamination trailer (MDT) prototype was tested through a series of three field trials. The first test of the MDT was conducted at Fort McClellan in Anniston, AL. during October 2004. Four test attempts occurred over two weekends; however, a number of system problems resulted in termination of the activity prior to any ClO{sub 2} introduction into the test building. After making several design enhancements and equipment changes, the MDT was subjected to a second test. During this test, extensive leak checks were made using argon and nitrogen in lieu of chlorine gas; each subsystem was checked for functionality, and the MDT was operated for 24 h. This second test demonstrated the MDT flow and control systems functioned satisfactorily, and thus it was decided to proceed to a third, more challenging field trial. In the last field test, ClO{sub 2} was generated and routed directly to the scrubber in a 12-h continuous run. Measurement of ClO{sub 2} levels at the generator outlet showed that the desired production rate was not achieved. Additionally, only one of the two scrubbers performed adequately with regard to maintaining ClO{sub 2} emissions below the limit. Numerous lessons were learned in the field trials of this ClO{sub 2} decontamination technology.

  20. Hfqs in Bacillus anthracis: Role of protein sequence variation in the structure and function of proteins in the Hfq family.

    Science.gov (United States)

    Vrentas, Catherine; Ghirlando, Rodolfo; Keefer, Andrea; Hu, Zonglin; Tomczak, Aurelie; Gittis, Apostolos G; Murthi, Athulaprabha; Garboczi, David N; Gottesman, Susan; Leppla, Stephen H

    2015-11-01

    Hfq proteins in Gram-negative bacteria play important roles in bacterial physiology and virulence, mediated by binding of the Hfq hexamer to small RNAs and/or mRNAs to post-transcriptionally regulate gene expression. However, the physiological role of Hfqs in Gram-positive bacteria is less clear. Bacillus anthracis, the causative agent of anthrax, uniquely expresses three distinct Hfq proteins, two from the chromosome (Hfq1, Hfq2) and one from its pXO1 virulence plasmid (Hfq3). The protein sequences of Hfq1 and 3 are evolutionarily distinct from those of Hfq2 and of Hfqs found in other Bacilli. Here, the quaternary structure of each B. anthracis Hfq protein, as produced heterologously in Escherichia coli, was characterized. While Hfq2 adopts the expected hexamer structure, Hfq1 does not form similarly stable hexamers in vitro. The impact on the monomer-hexamer equilibrium of varying Hfq C-terminal tail length and other sequence differences among the Hfqs was examined, and a sequence region of the Hfq proteins that was involved in hexamer formation was identified. It was found that, in addition to the distinct higher-order structures of the Hfq homologs, they give rise to different phenotypes. Hfq1 has a disruptive effect on the function of E. coli Hfq in vivo, while Hfq3 expression at high levels is toxic to E. coli but also partially complements Hfq function in E. coli. These results set the stage for future studies of the roles of these proteins in B. anthracis physiology and for the identification of sequence determinants of phenotypic complementation.

  1. Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Rosemary S Turingan

    Full Text Available BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed "Rapid Focused Sequencing," allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental

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

    Science.gov (United States)

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

    2015-01-01

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

  3. Ecological, Pedagogical, Public Rhetoric

    Science.gov (United States)

    Rivers, Nathaniel A.; Weber, Ryan P.

    2011-01-01

    Public rhetoric pedagogy can benefit from an ecological perspective that sees change as advocated not through a single document but through multiple mundane and monumental texts. This article summarizes various approaches to rhetorical ecology, offers an ecological read of the Montgomery bus boycotts, and concludes with pedagogical insights on a…

  4. Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

    Directory of Open Access Journals (Sweden)

    Ciervo Alessandra

    2006-04-01

    Full Text Available Abstract Background The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR Analysis (MLVA assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. Results Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. Conclusion In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the

  5. A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Denoeud France

    2001-03-01

    Full Text Available Abstract Background Some pathogenic bacteria are genetically very homogeneous, making strain discrimination difficult. In the last few years, tandem repeats have been increasingly recognized as markers of choice for genotyping a number of pathogens. The rapid evolution of these structures appears to contribute to the phenotypic flexibility of pathogens. The availability of whole-genome sequences has opened the way to the systematic evaluation of tandem repeats diversity and application to epidemiological studies. Results This report presents a database (http://minisatellites.u-psud.fr of tandem repeats from publicly available bacterial genomes which facilitates the identification and selection of tandem repeats. We illustrate the use of this database by the characterization of minisatellites from two important human pathogens, Yersinia pestis and Bacillus anthracis. In order to avoid simple sequence contingency loci which may be of limited value as epidemiological markers, and to provide genotyping tools amenable to ordinary agarose gel electrophoresis, only tandem repeats with repeat units at least 9 bp long were evaluated. Yersinia pestis contains 64 such minisatellites in which the unit is repeated at least 7 times. An additional collection of 12 loci with at least 6 units, and a high internal conservation were also evaluated. Forty-nine are polymorphic among five Yersinia strains (twenty-five among three Y. pestis strains. Bacillus anthracis contains 30 comparable structures in which the unit is repeated at least 10 times. Half of these tandem repeats show polymorphism among the strains tested. Conclusions Analysis of the currently available bacterial genome sequences classifies Bacillus anthracis and Yersinia pestis as having an average (approximately 30 per Mb density of tandem repeat arrays longer than 100 bp when compared to the other bacterial genomes analysed to date. In both cases, testing a fraction of these sequences for

  6. 炭疽杆菌鞭毛的初步研究%The Preliminary Studies on Flagellum of Bacillus anthracis

    Institute of Scientific and Technical Information of China (English)

    梁旭东

    1999-01-01

    通过半固体扩散生长法发现中国炭疽杆菌(Bacillus anthracis)大多数具有鞭毛,并经各种经典方法和分子生物学方法证实了这一结果,提示具有鞭毛是中国炭疽杆菌的一种特征,且不排除国外也有这种特征的菌株.

  7. 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.

  8. 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.

  9. Composite sampling of a Bacillus anthracis surrogate with cellulose sponge surface samplers from a nonporous surface.

    Directory of Open Access Journals (Sweden)

    Jenia A M Tufts

    Full Text Available A series of experiments was conducted to explore the utility of composite-based collection of surface samples for the detection of a Bacillus anthracis surrogate using cellulose sponge samplers on a nonporous stainless steel surface. Two composite-based collection approaches were evaluated over a surface area of 3716 cm2 (four separate 929 cm2 areas, larger than the 645 cm2 prescribed by the standard Centers for Disease Control (CDC and Prevention cellulose sponge sampling protocol for use on nonporous surfaces. The CDC method was also compared to a modified protocol where only one surface of the sponge sampler was used for each of the four areas composited. Differences in collection efficiency compared to positive controls and the potential for contaminant transfer for each protocol were assessed. The impact of the loss of wetting buffer from the sponge sampler onto additional surface areas sampled was evaluated. Statistical tests of the results using ANOVA indicate that the collection of composite samples using the modified sampling protocol is comparable to the collection of composite samples using the standard CDC protocol (p  =  0.261. Most of the surface-bound spores are collected on the first sampling pass, suggesting that multiple passes with the sponge sampler over the same surface may be unnecessary. The effect of moisture loss from the sponge sampler on collection efficiency was not significant (p  =  0.720 for both methods. Contaminant transfer occurs with both sampling protocols, but the magnitude of transfer is significantly greater when using the standard protocol than when the modified protocol is used (p<0.001. The results of this study suggest that composite surface sampling, by either method presented here, could successfully be used to increase the surface area sampled per sponge sampler, resulting in reduced sampling times in the field and decreased laboratory processing cost and turn-around times.

  10. Bacillus thuringiensis as a surrogate for Bacillus anthracis in aerosol research.

    Science.gov (United States)

    Tufts, Jenia A M; Calfee, M Worth; Lee, Sang Don; Ryan, Shawn P

    2014-05-01

    Characterization of candidate surrogate spores prior to experimental use is critical to confirm that the surrogate characteristics are as closely similar as possible to those of the pathogenic agent of interest. This review compares the physical properties inherent to spores of Bacillus anthracis (Ba) and Bacillus thuringiensis (Bt) that impact their movement in air and interaction with surfaces, including size, shape, density, surface morphology, structure and hydrophobicity. Also evaluated is the impact of irradiation on the physical properties of both Bacillus species. Many physical features of Bt and Ba have been found to be similar and, while Bt is considered typically non-pathogenic, it is in the B. cereus group, as is Ba. When cultured and sporulated under similar conditions, both microorganisms share a similar cylindrical pellet shape, an aerodynamic diameter of approximately 1 μm (in the respirable size range), have an exosporium with a hairy nap, and have higher relative hydrophobicities than other Bacillus species. While spore size, morphology, and other physical properties can vary among strains of the same species, the variations can be due to growth/sporulation conditions and may, therefore, be controlled. Growth and sporulation conditions are likely among the most important factors that influence the representativeness of one species, or preparation, to another. All Bt spores may, therefore, not be representative of all Ba spores. Irradiated spores do not appear to be a good surrogate to predict the behavior of non-irradiated spores due to structural damage caused by the irradiation. While the use of Bt as a surrogate for Ba in aerosol testing appears to be well supported, this review does not attempt to narrow selection between Bt strains. Comparative studies should be performed to test the hypothesis that viable Ba and Bt spores will behave similarly when suspended in the air (as an aerosol) and to compare the known microscale characteristics

  11. Screening of peptide libraries against protective antigen of Bacillus anthracis in a disposable microfluidic cartridge.

    Directory of Open Access Journals (Sweden)

    Joshua M Kogot

    Full Text Available Bacterial surface peptide display has gained popularity as a method of affinity reagent generation for a wide variety of applications ranging from drug discovery to pathogen detection. In order to isolate the bacterial clones that express peptides with high affinities to the target molecule, multiple rounds of manual magnetic activated cell sorting (MACS followed by multiple rounds of fluorescence activated cell sorting (FACS are conventionally used. Although such manual methods are effective, alternative means of library screening which improve the reproducibility, reduce the cost, reduce cross contamination, and minimize exposure to hazardous target materials are highly desired for practical application. Toward this end, we report the first semi-automated system demonstrating the potential for screening bacterially displayed peptides using disposable microfluidic cartridges. The Micro-Magnetic Separation platform (MMS is capable of screening a bacterial library containing 3 × 10¹⁰ members in 15 minutes and requires minimal operator training. Using this system, we report the isolation of twenty-four distinct peptide ligands that bind to the protective antigen (PA of Bacilus anthracis in three rounds of selection. A consensus motif WXCFTC was found using the MMS and was also found in one of the PA binders isolated by the conventional MACS/FACS approach. We compared MMS and MACS rare cell recovery over cell populations ranging from 0.1% to 0.0000001% and found that both magnetic sorting methods could recover cells down to 0.0000001% initial cell population, with the MMS having overall lower standard deviation of cell recovery. We believe the MMS system offers a compelling approach towards highly efficient, semi-automated screening of molecular libraries that is at least equal to manual magnetic sorting methods and produced, for the first time, 15-mer peptide binders to PA protein that exhibit better affinity and specificity than peptides

  12. Activated protein C ameliorates Bacillus anthracis lethal toxin-induced lethal pathogenesis in rats

    Directory of Open Access Journals (Sweden)

    Kau Jyh-Hwa

    2012-11-01

    Full Text Available Abstract Background Lethal toxin (LT is a major virulence factor of Bacillus anthracis. Sprague Dawley rats manifest pronounced lung edema and shock after LT treatments, resulting in high mortality. The heart failure that is induced by LT has been suggested to be a principal mechanism of lung edema and mortality in rodents. Since LT-induced death occurs more rapidly in rats than in mice, suggesting that other mechanisms in addition to the heart dysfunction may be contributed to the fast progression of LT-induced pathogenesis in rats. Coagulopathy may contribute to circulatory failure and lung injury. However, the effect of LT on coagulation-induced lung dysfunction is unclear. Methods To investigate the involvement of coagulopathy in LT-mediated pathogenesis, the mortality, lung histology and coagulant levels of LT-treated rats were examined. The effects of activated protein C (aPC on LT-mediated pathogenesis were also evaluated. Results Fibrin depositions were detected in the lungs of LT-treated rats, indicating that coagulation was activated. Increased levels of plasma D-dimer and thrombomodulin, and the ameliorative effect of aPC further suggested that the activation of coagulation-fibrinolysis pathways plays a role in LT-mediated pathogenesis in rats. Reduced mortality was associated with decreased plasma levels of D-dimer and thrombomodulin following aPC treatments in rats with LT-mediated pathogenesis. Conclusions These findings suggest that the activation of coagulation in lung tissue contributes to mortality in LT-mediated pathogenesis in rats. In addition, anticoagulant aPC may help to develop a feasible therapeutic strategy.

  13. Revisiting the Concept of Targeting Only Bacillus anthracis Toxins as a Treatment for Anthrax.

    Science.gov (United States)

    Glinert, Itai; Bar-David, Elad; Sittner, Assa; Weiss, Shay; Schlomovitz, Josef; Ben-Shmuel, Amir; Mechaly, Adva; Altboum, Zeev; Kobiler, David; Levy, Haim

    2016-08-01

    Protective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. The Bacillus anthracis toxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditions in vitro, may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets.

  14. Philosophy of ecology

    CERN Document Server

    Brown, Bryson; Peacock, Kent A

    2011-01-01

    The most pressing problems facing humanity today - over-population, energy shortages, climate change, soil erosion, species extinctions, the risk of epidemic disease, the threat of warfare that could destroy all the hard-won gains of civilization, and even the recent fibrillations of the stock market - are all ecological or have a large ecological component. in this volume philosophers turn their attention to understanding the science of ecology and its huge implications for the human project. To get the application of ecology to policy or other practical concerns right, humanity needs a clear and disinterested philosophical understanding of ecology which can help identify the practical lessons of science. Conversely, the urgent practical demands humanity faces today cannot help but direct scientific and philosophical investigation toward the basis of those ecological challenges that threaten human survival. This book will help to fuel the timely renaissance of interest in philosophy of ecology that is now oc...

  15. Human monoclonal antibodies against anthrax lethal factor and protective antigen act independently to protect against Bacillus anthracis infection and enhance endogenous immunity to anthrax

    NARCIS (Netherlands)

    Albrecht, Mark T.; Li, Han; Williamson, E. Diane; LeButt, Chris S.; Flick-Smith, Helen C.; Quinn, Conrad P.; Westra, Hans; Galloway, Darrell; Mateczun, Alfred; Goldman, Stanley; Groen, Herman; Baillie, Les W. J.

    2007-01-01

    The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action

  16. Bacillus anthracis acetyltransferases PatA1 and PatA2 modify the secondary cell wall polysaccharide and affect the assembly of S-layer proteins.

    Science.gov (United States)

    Lunderberg, J Mark; Nguyen-Mau, Sao-Mai; Richter, G Stefan; Wang, Ya-Ting; Dworkin, Jonathan; Missiakas, Dominique M; Schneewind, Olaf

    2013-03-01

    The envelope of Bacillus anthracis encompasses a proteinaceous S-layer with two S-layer proteins (Sap and EA1). Protein assembly in the envelope of B. anthracis requires S-layer homology domains (SLH) within S-layer proteins and S-layer-associated proteins (BSLs), which associate with the secondary cell wall polysaccharide (SCWP), an acetylated carbohydrate that is tethered to peptidoglycan. Here, we investigated the contributions of two putative acetyltransferases, PatA1 and PatA2, on SCWP acetylation and S-layer assembly. We show that mutations in patA1 and patA2 affect the chain lengths of B. anthracis vegetative forms and perturb the deposition of the BslO murein hydrolase at cell division septa. The patA1 and patA2 mutants are defective for the assembly of EA1 in the envelope but retain the ability of S-layer formation with Sap. SCWP isolated from the patA1 patA2 mutant lacked acetyl moieties identified in wild-type polysaccharide and failed to associate with the SLH domains of EA1. A model is discussed whereby patA1- and patA2-mediated acetylation of SCWP enables the deposition of EA1 as well as BslO near the septal region of the B. anthracis envelope.

  17. Identification of stringent response-related and potential serological proteins released from Bacillus anthracis overexpressing the RelA/SpoT homolog, Rsh Bant.

    Science.gov (United States)

    Kim, Se Kye; Park, Moon Kyoo; Kim, Sang Hoon; Oh, Kwang Gun; Jung, Kyoung Hwa; Hong, Chong-Hae; Yoon, Jang W; Chai, Young Gyu

    2014-10-01

    RelA and SpoT synthesize ppGpp, a key effector molecule that facilitates the adaptation of bacteria to nutrient starvation and other stresses, known as the stringent response. To investigate the role of Rsh Bant , a putative RelA/SpoT homolog (encoded by BAS4302) in Bacillus anthracis, we examined the alteration of the secretome profiles after the overexpression of a functional His-Rsh Bant protein in the B. anthracis strain Sterne at the stationary growth phase. In the ppGpp-deficient E. coli mutant strain CF1693, overexpression of Rsh Bant restored a ppGpp-dependent growth defect on minimal glucose media. The secretome profiles obtained using a two-dimensional electrophoresis (2-DE) analysis were altered by overexpression of Rsh Bant in B. anthracis. Among the 66 protein spots differentially expressed >1.5-fold, the 29 proteins were abundant for further identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Functional categorization of those proteins implicated their involvement in various biological activities. Taken together, our results imply that overexpression of a functional His-Rsh Bant can lead to the increased levels of intracellular ppGpp in B. anthracis, resulting in the significant changes in its secretome profiling. The stringent response-controlled proteins identified are likely useful as potential targets for serodiagnostic applications.

  18. Delineating the effect of host environmental signals on a fully virulent strain of Bacillus anthracis using an integrated transcriptomics and proteomics approach

    NARCIS (Netherlands)

    Panda, G.; Basak, T.; Tanwer, P.; Sengupta, S.; Martins dos Santos, V.A.P.; Bhatnagar, R.

    2014-01-01

    Pathogenic bacteria sense the host environment and regulate expression of virulence-related genes. Environmental signals like temperature, bicarbonate/CO2 and glucose induce toxin production in Bacillus anthracis, but the mechanisms by which these signals contribute to virulence and overall physiolo

  19. Virtual screening of LPXTG competitive SrtA inhibitors targeting signal transduction mechanism in Bacillus anthracis: a combined experimental and theoretical study.

    Science.gov (United States)

    Selvaraj, Chandrabose; Sivakamavalli, Jeyachandran; Baskaralingam, Vaseeharan; Singh, Sanjeev Kumar

    2014-06-01

    Members of the sortase enzyme super family decorate the surfaces of Bacillus anthracis cell wall with proteins that play key roles in microbial pathogenesis and its biofilm formation. Bacillus anthracis Sortase-A (Ba-SrtA) is a potential target for new therapeutics as it is required for B. anthracis survival and replication within macrophages. An understanding of the binding site pocket and substrate recognition mechanism by SrtA enzymes may serve to be beneficial in the rational development of sortase inhibitors. Here, the LPXTG signal peptide-based competitive inhibitors are screened against the Ba-SrtA and compounds with reasonable inhibition, specificity, and mechanisms of inactivation of SrtA have been covered. The screened compounds are experimentally validated against the phylogenetically similar Gram-positive pathogen B. cereus. In situ microscopic visualizations suggest that these screened compounds showed the microbial and biofilm inhibitory activity against B. cereus. It facilitates the further development of these molecules into useful anti-infective agents to treat infections caused by B. anthracis and other Gram-positive pathogens. These results provide insight into basic design principles for generating new clinically relevant lead molecules. It also provides an alternative strategy where a screened ligand molecule can be used in combination to battle increasingly against the Gram-positive pathogens.

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

    NARCIS (Netherlands)

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

    2013-01-01

    Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory- based matrix-assisted laser desorptio

  1. Identification of B. anthracis N(5)-carboxyaminoimidazole ribonucleotide mutase (PurE) active site binding compounds via fragment library screening.

    Science.gov (United States)

    Lei, Hao; Jones, Christopher; Zhu, Tian; Patel, Kavankumar; Wolf, Nina M; Fung, Leslie W-M; Lee, Hyun; Johnson, Michael E

    2016-02-15

    The de novo purine biosynthesis pathway is an attractive target for antibacterial drug design, and PurE from this pathway has been identified to be crucial for Bacillus anthracis survival in serum. In this study we adopted a fragment-based hit discovery approach, using three screening methods-saturation transfer difference nucleus magnetic resonance (STD-NMR), water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR, and surface plasmon resonance (SPR), against B. anthracis PurE (BaPurE) to identify active site binding fragments by initially testing 352 compounds in a Zenobia fragment library. Competition STD NMR with the BaPurE product effectively eliminated non-active site binding hits from the primary hits, selecting active site binders only. Binding affinities (dissociation constant, KD) of these compounds varied between 234 and 301μM. Based on test results from the Zenobia compounds, we subsequently developed and applied a streamlined fragment screening strategy to screen a much larger library consisting of 3000 computationally pre-selected fragments. Thirteen final fragment hits were confirmed to exhibit binding affinities varying from 14μM to 700μM, which were categorized into five different basic scaffolds. All thirteen fragment hits have ligand efficiencies higher than 0.30. We demonstrated that at least two fragments from two different scaffolds exhibit inhibitory activity against the BaPurE enzyme.

  2. Bacillus anthracis lethal toxin disrupts TCR signaling in CD1d-restricted NKT cells leading to functional anergy.

    Directory of Open Access Journals (Sweden)

    Sunil K Joshi

    2009-09-01

    Full Text Available Exogenous CD1d-binding glycolipid (alpha-Galactosylceramide, alpha-GC stimulates TCR signaling and activation of type-1 natural killer-like T (NKT cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA-mediated intracellular delivery of lethal factor (LF, a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8 and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

  3. Assessment of delivery parameters with the multi-electrode array for development of a DNA vaccine against Bacillus anthracis.

    Science.gov (United States)

    Donate, Amy; Heller, Richard

    2013-12-01

    Gene electrotransfer (GET) enhances delivery of DNA vaccines by increasing both gene expression and immune responses. Our lab has developed the multi-electrode array (MEA) for DNA delivery to skin. The MEA was used at constant pulse duration (150 ms) and frequency (6.67 Hz). In this study, delivery parameters including applied voltage (5-45 V), amount of plasmid (100-300 μg), and number of treatments (2-3) were evaluated for delivery of a DNA vaccine. Mice were intradermally injected with plasmid expressing Bacillus anthracis protective antigen with or without GET and αPA serum titers measured. Within this experiment no significant differences were noted in antibody levels from varying dose or treatment number. However, significant differences were measured from applied voltages of 25 and 35 V. These voltages generated antibody levels between 20,000 and 25,000. Serum from animals vaccinated with these conditions also resulted in toxin neutralization in 40-60% of animals. Visual damage was noted at MEA conditions of 40 V. No damage was noted either visually or histologically from conditions of 35 V or below. These results reflect the importance of establishing appropriate electrical parameters and the potential for the MEA in non-invasive DNA vaccination against B. anthracis.

  4. What is dental ecology?

    Science.gov (United States)

    Cuozzo, Frank P; Sauther, Michelle L

    2012-06-01

    Teeth have long been used as indicators of primate ecology. Early work focused on the links between dental morphology, diet, and behavior, with more recent years emphasizing dental wear, microstructure, development, and biogeochemistry, to understand primate ecology. Our study of Lemur catta at the Beza Mahafaly Special Reserve, Madagascar, has revealed an unusual pattern of severe tooth wear and frequent tooth loss, primarily the result of consuming a fallback food for which these primates are not dentally adapted. Interpreting these data was only possible by combining our areas of expertise (dental anatomy [FC] and primate ecology [MS]). By integrating theoretical, methodological, and applied aspects of both areas of research, we adopted the term "dental ecology"-defined as the broad study of how teeth respond to the environment. Specifically, we view dental ecology as an interpretive framework using teeth as a vehicle for understanding an organism's ecology, which builds upon earlier work, but creates a new synthesis of anatomy and ecology that is only possible with detailed knowledge of living primates. This framework includes (1) identifying patterns of dental pathology and tooth use-wear, within the context of feeding ecology, behavior, habitat variation, and anthropogenic change, (2) assessing ways in which dental development and biogeochemical signals can reflect habitat, environmental change and/or stress, and (3) how dental microstructure and macro-morphology are adapted to, and reflect feeding ecology. Here we define dental ecology, provide a short summary of the development of this perspective, and place our new work into this context.

  5. Microevolution of Anthrax from a Young Ancestor (M.A.Y.A. Suggests a Soil-Borne Life Cycle of Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Peter Braun

    Full Text Available During an anthrax outbreak at the Pollino National Park (Basilicata, Italy in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA and next generation Whole Genome Sequencing (WGS. PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis.

  6. Microevolution of Anthrax from a Young Ancestor (M.A.Y.A.) Suggests a Soil-Borne Life Cycle of Bacillus anthracis.

    Science.gov (United States)

    Braun, Peter; Grass, Gregor; Aceti, Angela; Serrecchia, Luigina; Affuso, Alessia; Marino, Leonardo; Grimaldi, Stefania; Pagano, Stefania; Hanczaruk, Matthias; Georgi, Enrico; Northoff, Bernd; Schöler, Anne; Schloter, Michael; Antwerpen, Markus; Fasanella, Antonio

    2015-01-01

    During an anthrax outbreak at the Pollino National Park (Basilicata, Italy) in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA) and next generation Whole Genome Sequencing (WGS). PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis.

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

    Science.gov (United States)

    Addae, Ebenezer

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

  8. Failure of Sterne- and Pasteur-like strains of Bacillus anthracis to replicate and survive in the urban bluebottle blow fly Calliphora vicina under laboratory conditions.

    Directory of Open Access Journals (Sweden)

    Britta von Terzi

    Full Text Available This study aimed to elucidate the bacteriological events occurring within the gut of Calliphora vicina, selected as the European representative of blow flies held responsible for the spread of anthrax during epidemics in certain parts of the world. Green-fluorescent-protein-carrying derivatives of Bacillus anthracis were used. These lacked either one of the virulence plasmids pXO1 and pXO2 and were infected, or not infected, with a worm intestine phage (Wip4 known to influence the phenotype and survival of the pathogen. Blood meals were prepared for the flies by inoculation of sheep blood with germinated and, in case of pXO2+ strains, encapsulated cells of the four B. anthracis strains. After being fed for 4 h an initial 10 flies were externally disinfected with peracetic acid to ensure subsequent quantitation representing ingested B. anthracis only. Following neutralization, they were crushed in sterile saline. Over each of the ensuing 7 to 10 days, 10 flies were removed and processed the same way. In the absence of Wip4, strains showed steady declines to undetectable in the total B. anthracis counts, within 7-9 days. With the phage infected strains, the falls in viable counts were significantly more rapid than in their uninfected counterparts. Spores were detectable in flies for longer periods than vegetative bacteria. In line with the findings in both biting and non-biting flies of early workers our results indicate that B. anthracis does not multiply in the guts of blow flies and survival is limited to a matter of days.

  9. Development of Ecological Buildings

    Directory of Open Access Journals (Sweden)

    Andrius Keizikas

    2011-04-01

    Full Text Available The article presents research on ecological buildings and their influence on the constructional sphere. The aim of the paper is to reveal the essence of ecological architecture showing substantial progress and its potential to stimulate architectural and technological growth. The article also describes relations between the ideas of ecological buildings and the ‘passive house’ concepts and aspects of development as well as describes the possibilities of improving building sustainability and energy efficiency. Article in Lithuanian

  10. Social Ecological Economics

    OpenAIRE

    Spash, Clive L.

    2009-01-01

    This paper introduces and explains how ecological economics has developed as a modern movement with its roots in environmentalism and radical environmental economics. Divisions and conflicts within the field are explored to show why material claiming to fall under the title of ecological economics fails to be representative of progress or the vision which drove socio-economic specialists to interact with ecologists in the first place. The argument is then put forward that ecological economics...

  11. Between Design and Ecology

    DEFF Research Database (Denmark)

    Bjørn, Mona Chor

    such vegetation, based on concepts and theories in plant community ecology. If these communities are based on local forbs there is a continuum in anthropogenic intervention from designed and intensively maintained to semi-natural herbaceous vegetation. Results from a large field experiment show that, after three...... colonised by grasses and eventually woody species. This thesis adds useful basic knowledge in plant community ecology and species-specific growth, which are relevant to research and planning in landscape architecture and ecology....

  12. [Demography and human ecology].

    Science.gov (United States)

    Nazareth, J M

    1993-01-01

    At the end of the 19th century the German biologist Ernest Haekel was the first scientist to use the term ecology, which was defined as the study of relationships of organisms or groups of organisms with the environment and indicated the interdependence of the living world, including plants, animals, and humans. This concept also indicates a continuous process of adaptation of organisms to their external environment. The basic concepts of scientific ecology, which developed at the end of the 19th century, can be attributed to Darwin: the relationships between living beings and the notion of the process of adaptation to their environment. The term human ecology appeared in the early 1920s. Human ecology embodies fundamental ideas: biotype, habitat, community, biocenosis, ecosystem, biomass, interchange and equilibrium, and circulation of energy. The accumulated knowledge about human ecology is broken down using the criteria of topography (ecology of humid forests, deserts, lakes, etc.); followed by the appearance of species; and the variants of classical division: auto ecology (influence of external factors on living beings) and sinecology (the study of groups of associated organisms, i.e., natural, animal, and vegetation communities). The species are considered on the basis of equality or sinecology (all of them have the same interests), while in human ecology a species is determined by its relation to a reference group--autoecology or anthropocentric ecology. In 1911, J. Thompson bridged the gap between biological knowledge and social sciences; in 1921, H. Barrows identified human ecology as a component of geography; in 1925, L. Bernard presented the classification of ecosystems; and in 1936, Ezra Park published his work, Human Ecology, followed in 1945 by the emergence of the Chicago school. Demography and human ecology are intimately connected because population is the result of natural and migratory movements, therefore the two sciences require a methodology

  13. Chemical ecology of fungi.

    Science.gov (United States)

    Spiteller, Peter

    2015-07-01

    Fungi are widespread in nature and have conquered nearly every ecological niche. Fungi occur not only in terrestrial but also in freshwater and marine environments. Moreover, fungi are known as a rich source of secondary metabolites. Despite these facts, the ecological role of many of these metabolites is still unknown and the chemical ecology of fungi has not been investigated systematically so far. This review intends to present examples of the various chemical interactions of fungi with other fungi, plants, bacteria and animals and to give an overview of the current knowledge of fungal chemical ecology.

  14. CD4+ T cells targeting dominant and cryptic epitopes from Bacillus anthracis Lethal Factor

    Directory of Open Access Journals (Sweden)

    Stephanie eAscough

    2016-01-01

    Full Text Available Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called ‘cryptic’ or ‘subdominant’ epitopes. We analysed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISPOT assays we characterised epitopes that elicited a response following immunisation with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 trangenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were

  15. Naturally acquired antibodies to Bacillus anthracis protective antigen in vultures of southern Africa

    Directory of Open Access Journals (Sweden)

    P. C.B. Turnbull

    2008-08-01

    Full Text Available TURNBULLP, P.C.B. DIEKMANNM,M., KILIAN, J.W., VERSFELDW, W.,DE VOS, V., ARNTZENL, L.,WOLTER, K., BARTELS, P. & KOTZE, A. 2008.N aturally acquired antibodies to Bacillusa nthracisp rotective antigeni n vultureso f southern Africa. Onderstepoort Journal of Veterinary Research, T5:95-102 Sera from 19 wild caught vultures in northern Namibia and 15 (12 wild caught and three captive bred but with minimal histories in North West Province, South Africa, were examined by an enzyme-linked immunosorbenats say( ELISAf or antibodiesto the Bacillus anthracis toxin protective antigen (PA. As assessed from the baseline established with a control group of ten captive reared vultures with well-documented histories, elevated titres were found in 12 of the 19 (63% wild caught Namibian birds as compared with none of the 15 South African ones. There was a highly significant difference between the Namibian group as a hole and the other groups (P < 0.001 and no significant difference between the South African and control groups (P > 0.05. Numbers in the Namibian group were too small to determine any significances in species-, sex- or age-related differences within the raw data showing elevated titres in four out of six Cape Vultures, Gyps coprotheress, six out of ten Whitebacked Vultures, Gyps africanus, and one out of three Lappet-faced Vultures, Aegypiust racheliotus, or in five of six males versus three of seven females, and ten of 15 adults versus one of four juveniles. The results are in line with the available data on the incidence of anthrax in northern Namibia and South Africa and the likely contact of the vultures tested with anthrax carcasses. lt is not known whether elevated titre indicates infection per se in vultures or absorption of incompletely digested epitopes of the toxin or both. The results are discussed in relation to distances travelled by vultures as determined by new tracking techniques, how serology can reveal anthrax activity in an area and

  16. 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

  17. Identification of Novel Raft Marker Protein, FlotP in Bacillus anthracis.

    Science.gov (United States)

    Somani, Vikas K; Aggarwal, Somya; Singh, Damini; Prasad, Tulika; Bhatnagar, Rakesh

    2016-01-01

    Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP) harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB) also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homolog harboring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, four were found to harbor RMP. Among them, domains of Bas0525 (FlotP) of Bacillus anthracis (BA) showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA), a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown raft associated

  18. Bacteriophage-mediated acquisition of antibiotic resistance by Staphylococcus aureus type 88.

    OpenAIRE

    Schaefler, S.

    1982-01-01

    Antibiotic-resistant Staphylococcus aureus strains of phage type 88, lysogenic for phage 188, when grown in mixed culture with a nonlysogenic novobiocin-resistant strain, acquired novobiocin resistance at a high rate from the nonlysogenic strain. With most strains of phage type 88, there was no detectable transfer of resistance from lysogenic to nonlysogenic cells. Lysogenization with phage 188 of phage-sensitive strains conferred on the lysogenized cells the ability to acquire chromosome and...

  19. Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents

    Directory of Open Access Journals (Sweden)

    Amanda Donnelly

    2015-12-01

    Full Text Available The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i provides enhanced phage-mediated gene transfer; (ii is applicable for laboratory transfection processes and (iii shows promise within industry for large-scale gene transfer applications.

  20. Possession, Use, and Transfer of Select Agents and Toxins-- Addition of Bacillus Cereus Biovar Anthracis to the HHS List of Select Agents and Toxins. Interim final rule and request for comments.

    Science.gov (United States)

    2016-09-14

    The Centers for Disease Control and Prevention (CDC) in the Department of Health and Human Services (HHS) is adding Bacillus cereus Biovar anthracis to the list of HHS select agents and toxins as a Tier 1 select agent. We are taking this action to regulate this agent that is similar to B. anthracis to prevent its misuse, which could cause a biological threat to public health and/or national security.

  1. Ecologies of Learning, Ecologies of Creativity

    DEFF Research Database (Denmark)

    Høyrup, Helene

    The subject of this project/paper is the challenges that digital media pose to earlier definitions, divisions and conceptions of learning institutions and their activities. It is argued that, in the light of digital media, the idea of institutional co-creation of learning and literacies needs...... in the light of the new Danish school reform. How can different learning institutions contribute to a “joint” ecology of learning? What would the benefits be from this in terms of young people’s literacies? On what theoretical basis can such an ecology and co-creation take place? And what kind of didactics...

  2. Dynamics in artifact ecologies

    DEFF Research Database (Denmark)

    Bødker, Susanne; Klokmose, Clemens Nylandsted

    2012-01-01

    artifacts influence the use of others. Understanding this interplay becomes more and more essential for interaction design as our artifact ecologies grow. This paper continues a recent discourse on artifact ecologies. Through interviews with iPhone users, we demonstrate that relationships between artifacts...

  3. RADIOECOLOGY AND ECOLOGICAL IMMUNOLOGY

    Directory of Open Access Journals (Sweden)

    V. M. Shubik

    2009-01-01

    Full Text Available The author's investigations results are presented in comparing with literary materials concerning the application of principles and methods of ecological immunology for solving radioecological questions. The data on characteristic of immunity and health of human population affected with radiation factors of the environment is given as well as animals' population state as the links offood ecological chains.

  4. Quantitative plant ecology

    DEFF Research Database (Denmark)

    Damgaard, Christian

    2014-01-01

    This e-book is written in the Wolfram' CDF format (download free CDF player from Wolfram.com) The objective of this e-book is to introduce the population ecological concepts for measuring and predicting the ecological success of plant species. This will be done by focusing on the measurement......, will not be covered in this e-book....

  5. The ecology of citizenship

    NARCIS (Netherlands)

    Coates, Robert; Garmany, Jeff

    2017-01-01

    This article calls into question the relationship between citizenship, space and ecological stability. Drawing on case study research from urban Brazil, we argue that while space may be crucial to Western perspectives of citizenship – particularly in urban areas – the ecological coproduction of thes

  6. Predictive systems ecology.

    Science.gov (United States)

    Evans, Matthew R; Bithell, Mike; Cornell, Stephen J; Dall, Sasha R X; Díaz, Sandra; Emmott, Stephen; Ernande, Bruno; Grimm, Volker; Hodgson, David J; Lewis, Simon L; Mace, Georgina M; Morecroft, Michael; Moustakas, Aristides; Murphy, Eugene; Newbold, Tim; Norris, K J; Petchey, Owen; Smith, Matthew; Travis, Justin M J; Benton, Tim G

    2013-11-22

    Human societies, and their well-being, depend to a significant extent on the state of the ecosystems that surround them. These ecosystems are changing rapidly usually in response to anthropogenic changes in the environment. To determine the likely impact of environmental change on ecosystems and the best ways to manage them, it would be desirable to be able to predict their future states. We present a proposal to develop the paradigm of predictive systems ecology, explicitly to understand and predict the properties and behaviour of ecological systems. We discuss the necessary and desirable features of predictive systems ecology models. There are places where predictive systems ecology is already being practised and we summarize a range of terrestrial and marine examples. Significant challenges remain but we suggest that ecology would benefit both as a scientific discipline and increase its impact in society if it were to embrace the need to become more predictive.

  7. 炭疽芽孢杆菌S-层蛋白功能研究进展%S-layer proteins of Bacillus anthracis: research progress

    Institute of Scientific and Technical Information of China (English)

    王雪芳; 刘先凯; 陈福生; 王恒樑

    2011-01-01

    Bacillus anthracis is the etiological agent of anthrax. Currently, plasmids pXOl and pX02 of B. anthracis, which were encoding the virulence genes and capsule synthesis genes respectively, have been studied thoroughly. However, the S-layer, a protein paracrystalline structure that exists between the capsule and the cell wall of B. anthracis, is less known. Sap( surface array protein ) and EA1( extracellular antigen 1 ) are the major proteins of S-layer as well as some other relative proteins in B. anthracis. Investigation of the interaction of these proteins and their immunologic mechanism is vital for understanding the pathogenesis of B. anthracis. This brief review will focus on recent researches on the components of S-layer proteins, their linkage with the cell wall and the regulation patterns of their encoding genes of S-layer proteins, their role in the pathogenesis of B. anthracis, and the relationship between S-layer proteins and host immune systems.%炭疽芽孢杆菌是人畜共患病炭疽的病原菌.目前,对炭疽杆菌2个毒力大质粒(编码主要毒力基因的pXO1与编码合成荚膜基因的pXO2)的研究比较深入;炭疽杆菌细胞壁与荚膜间还存在一种蛋白性质的类晶体(paracrystalline)结构:S-层(surface-layer,表层)结构.炭疽杆菌的S-层蛋白主要为表面排列蛋白(surface array protein,Sap)和胞外抗原1(extracellular antigen 1,EA1),此外,在炭疽杆菌中还存在其他一些与S-层相关的蛋白,了解这些蛋白的相互作用及免疫机制对深入认识炭疽杆菌的致病机制具有重要意义.本文将就近年来关于炭疽杆菌S-层蛋白成分、与细胞壁的连接、S-层基因的调控、致病性及其与宿主免疫机制的关系等方面的研究进展做一简要综述.

  8. Evaluation of New Dihydrophthalazine-Appended 2,4-Diaminopyrimidines against Bacillus anthracis: Improved Syntheses Using a New Pincer Complex

    Directory of Open Access Journals (Sweden)

    Nagendra Prasad Muddala

    2015-04-01

    Full Text Available The synthesis and evaluation of ten new dihydrophthalazine-appended 2,4-diaminopyrimidines as potential drugs to treat Bacillus anthracis is reported. An improved synthesis utilizing a new pincer catalyst, dichlorobis[1-(dicyclohexylphosphanyl-piperidine]palladium(II, allows the final Heck coupling to be performed at 90 °C using triethylamine as the base. These milder conditions have been used to achieve improved yields for new and previously reported substrates with functional groups that degrade or react at the normal 140 °C reaction temperature. An analytical protocol for separating the S and R enantiomers of two of the most active compounds is also disclosed. Finally, the X-ray structure for the most active enantiomer of the lead compound, (S-RAB1, is given.

  9. Evaluation of Immunoassays and General Biological Indicator Tests for Field Screening of Bacillus anthracis and Ricin

    Energy Technology Data Exchange (ETDEWEB)

    Bartholomew, Rachel A.; Ozanich, Richard M.; Arce, Jennifer S.; Engelmann, Heather E.; Heredia-Langner, Alejandro; Hofstad, Beth A.; Hutchison, Janine R.; Jarman, Kristin; Melville, Angela M.; Victry, Kristin D.; Bruckner-Lea, Cynthia J.

    2017-02-01

    The goal of this testing was to evaluate the ability of currently available commercial off-the-shelf (COTS) biological indicator tests and immunoassays to detect Bacillus anthracis (Ba) spores and ricin. In general, immunoassays provide more specific identification of biological threats as compared to indicator tests [3]. Many of these detection products are widely used by first responders and other end users. In most cases, performance data for these instruments are supplied directly from the manufacturer, but have not been verified by an external, independent assessment [1]. Our test plan modules included assessments of inclusivity (ability to generate true positive results), commonly encountered hoax powders (which can cause potential interferences or false positives), and estimation of limit of detection (LOD) (sensitivity) testing.

  10. A strain-variable bacteriocin in Bacillus anthracis and Bacillus cereus with repeated Cys-Xaa-Xaa motifs

    Directory of Open Access Journals (Sweden)

    Haft Daniel H

    2009-04-01

    Full Text Available Abstract Bacteriocins are peptide antibiotics from ribosomally translated precursors, produced by bacteria often through extensive post-translational modification. Minimal sequence conservation, short gene lengths, and low complexity sequence can hinder bacteriocin identification, even during gene calling, so they are often discovered by proximity to accessory genes encoding maturation, immunity, and export functions. This work reports a new subfamily of putative thiazole-containing heterocyclic bacteriocins. It appears universal in all strains of Bacillus anthracis and B. cereus, but has gone unrecognized because it is always encoded far from its maturation protein operon. Patterns of insertions and deletions among twenty-four variants suggest a repeating functional unit of Cys-Xaa-Xaa. Reviewers This article was reviewed by Andrei Osterman and Lakshminarayan Iyer.

  11. 炭疽芽胞杆菌的基因分型%The genotyping of Bacillus anthracis

    Institute of Scientific and Technical Information of China (English)

    周伟(综述); 郭学军(审校)

    2013-01-01

    In order to identify the genotype of the Bacillus anthracis,multi-locus variable number tandem repeat analysis (MLVA), single nucleotide polymorphism analysis (SNP) and single nucleotide repeat analysis (SNR) have been used, and become the main methods gradually .This paper reviewes the status of their application .%为了鉴别炭疽芽胞杆菌( Bacillus anthracis)的基因型,多位点可变数量串联重复序列分析( MLVA)、单核苷酸多态性分析( SNP )和单核苷酸重复序列分析( SNR )被广泛应用,并逐渐成为炭疽分型的基本方法,因而对其应用现状进行了综述。

  12. Evaluation of the FilmArray® system for detection of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

    Energy Technology Data Exchange (ETDEWEB)

    Seiner, Derrick R.; Colburn, Heather A.; Baird, Cheryl L.; Bartholomew, Rachel A.; Straub, Tim M.; Victry, Kristin D.; Hutchison, Janine R.; Valentine, Nancy B.; Bruckner-Lea, Cindy J.

    2013-04-29

    To evaluate the sensitivity and specificity of the Idaho Technologies FilmArray® Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft), and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores. Methods and Results: DNA samples from Ba, Ft and Yp strains and near-neighbors, and live Ba spores were analyzed using the Biothreat Panel, a multiplexed PCR-based assay for 17 pathogens and toxins. Sensitivity studies with DNA suggest a limit of detection of 250 genome equivalents (GEs) per sample. Furthermore, the correct call of Ft, Yp or Bacillus species was made in 63 of 72 samples tested at 25 GE or less. With samples containing 25 Ba Sterne spores, at least one of the two possible Ba markers were identified in all samples tested. We observed no cross-reactivity with near-neighbor DNAs.

  13. The central nervous system as target of Bacillus anthracis toxin independent virulence in rabbits and guinea pigs.

    Science.gov (United States)

    Levy, Haim; Glinert, Itai; Weiss, Shay; Bar-David, Elad; Sittner, Assa; Schlomovitz, Josef; Altboum, Zeev; Kobiler, David

    2014-01-01

    Infection of the central nervous system is considered a complication of Anthrax and was reported in humans and non-human primates. Previously we have reported that Bacillus anthracis possesses a toxin-independent virulent trait that, like the toxins, is regulated by the major virulence regulator, AtxA, in the presence of pXO2. This toxin-independent lethal trait is exhibited in rabbits and Guinea pigs following significant bacteremia and organ dissemination. Various findings, including meningitis seen in humans and primates, suggested that the CNS is a possible target for this AtxA-mediated activity. In order to penetrate into the brain tissue, the bacteria have to overcome the barriers isolating the CNS from the blood stream. Taking a systematic genetic approach, we compared intracranial (IC) inoculation and IV/SC inoculation for the outcome of the infection in rabbits/GP, respectively. The outstanding difference between the two models is exhibited by the encapsulated strain VollumΔpXO1, which is lethal when injected IC, but asymptomatic when inoculated IV/SC. The findings demonstrate that there is an apparent bottleneck in the ability of mutants to penetrate into the brain. Any mutant carrying either pXO1 or pXO2 will kill the host upon IC injection, but only those carrying AtxA either on pXO1 or in the chromosome in the background of pXO2 can penetrate into the brain following peripheral inoculation. The findings were corroborated by histological examination by H&E staining and immunofluorescence of rabbits' brains following IV and IC inoculations. These findings may have major implications on future research both on B. anthracis pathogenicity and on vaccine development.

  14. The central nervous system as target of Bacillus anthracis toxin independent virulence in rabbits and guinea pigs.

    Directory of Open Access Journals (Sweden)

    Haim Levy

    Full Text Available Infection of the central nervous system is considered a complication of Anthrax and was reported in humans and non-human primates. Previously we have reported that Bacillus anthracis possesses a toxin-independent virulent trait that, like the toxins, is regulated by the major virulence regulator, AtxA, in the presence of pXO2. This toxin-independent lethal trait is exhibited in rabbits and Guinea pigs following significant bacteremia and organ dissemination. Various findings, including meningitis seen in humans and primates, suggested that the CNS is a possible target for this AtxA-mediated activity. In order to penetrate into the brain tissue, the bacteria have to overcome the barriers isolating the CNS from the blood stream. Taking a systematic genetic approach, we compared intracranial (IC inoculation and IV/SC inoculation for the outcome of the infection in rabbits/GP, respectively. The outstanding difference between the two models is exhibited by the encapsulated strain VollumΔpXO1, which is lethal when injected IC, but asymptomatic when inoculated IV/SC. The findings demonstrate that there is an apparent bottleneck in the ability of mutants to penetrate into the brain. Any mutant carrying either pXO1 or pXO2 will kill the host upon IC injection, but only those carrying AtxA either on pXO1 or in the chromosome in the background of pXO2 can penetrate into the brain following peripheral inoculation. The findings were corroborated by histological examination by H&E staining and immunofluorescence of rabbits' brains following IV and IC inoculations. These findings may have major implications on future research both on B. anthracis pathogenicity and on vaccine development.

  15. Two-component system cross-regulation integrates Bacillus anthracis response to heme and cell envelope stress.

    Directory of Open Access Journals (Sweden)

    Laura A Mike

    2014-03-01

    Full Text Available Two-component signaling systems (TCSs are one of the mechanisms that bacteria employ to sense and adapt to changes in the environment. A prototypical TCS functions as a phosphorelay from a membrane-bound sensor histidine kinase (HK to a cytoplasmic response regulator (RR that controls target gene expression. Despite significant homology in the signaling domains of HKs and RRs, TCSs are thought to typically function as linear systems with little to no cross-talk between non-cognate HK-RR pairs. Here we have identified several cell envelope acting compounds that stimulate a previously uncharacterized Bacillus anthracis TCS. Furthermore, this TCS cross-signals with the heme sensing TCS HssRS; therefore, we have named it HssRS interfacing TCS (HitRS. HssRS reciprocates cross-talk to HitRS, suggesting a link between heme toxicity and cell envelope stress. The signaling between HssRS and HitRS occurs in the parental B. anthracis strain; therefore, we classify HssRS-HitRS interactions as cross-regulation. Cross-talk between HssRS and HitRS occurs at both HK-RR and post-RR signaling junctions. Finally, HitRS also regulates a previously unstudied ABC transporter implicating this transporter in the response to cell envelope stress. This chemical biology approach to probing TCS signaling provides a new model for understanding how bacterial signaling networks are integrated to enable adaptation to complex environments such as those encountered during colonization of the vertebrate host.

  16. Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity.

    Science.gov (United States)

    Hammerstrom, Troy G; Horton, Lori B; Swick, Michelle C; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M

    2015-02-01

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO(2)/bicarbonate, and there is a positive correlation between the CO(2)/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His→Asp) and phosphoablative (His→Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.

  17. Comparison of Packed Beds and Qiagen Columns for Recovering Trace Amounts of B. anthracis DNA from Liquid Suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Sorensen, K; Arroyo, E; Erler, A; Christian, A T; Camp, D; Wheeler, E K

    2006-06-23

    The goal of this work was to optimize and evaluate LLNL's in-bed amplification technology to improve the level of detection for suspensions containing trace amounts of anthracis DNA. The binding/cleaning performance of the packed bed is compared to the conventional commercial approach; Qiagen column cleanup and elution, followed by detection through an ex-situ amplification process. Five liquid suspensions were spiked with B.anthracis DNA in concentration series. These suspensions were: (1) water, (2) water with EDTA, (3) dirty water from carpet extraction, (4) dirty carpet extraction with phosphate buffered saline (PBS) plus 0.1% Tween 20 plus 0.1% gelatin, and (5) a subway aerosol collected in water. Each suspension matrix was spiked with DNA and injected (in replicate) into either Qiagen Microcolumns (using the kit processing instructions) or LLNL's packed bed (using the LLNL in-bed purification and amplification protocol). The process output was assayed by quantitative polymerase chain reaction (QPCR). Table ES-1 shows the level of DNA (pg per 100 uL of input suspension) that resulted in successful amplification for all reactions (X=Y), and the level for which at least one of the reactions was successful (X>0). For each suspension and DNA concentration, there were Y QPCR assays of which X showed successful amplification. LLNL's packed bed technology outperformed Qiagen Microcolumns for all five suspensions, typically by one order of magnitude in both the limit of assured detection (all reactions positive), and the lower limit of detection (some reactions positive).

  18. Detection probability models for bacteria, and how to obtain them from heterogeneous spiking data. An application to Bacillus anthracis.

    Science.gov (United States)

    Hedell, Ronny; Stephansson, Olga; Mostad, Petter; Andersson, Mats Gunnar

    2017-01-16

    Efficient and correct evaluation of sampling results with respect to hypotheses about the concentration or distribution of bacteria generally requires knowledge about the performance of the detection method. To assess the sensitivity of the detection method an experiment is usually performed where the target matrix is spiked (i.e. artificially contaminated) with different concentrations of the bacteria, followed by analyses of the samples using the pre-enrichment method and the analytical detection method of interest. For safety reasons or because of economic or time limits it is not always possible to perform exactly such an experiment, with the desired number of samples. In this paper, we show how heterogeneous data from diverse sources may be combined within a single model to obtain not only estimates of detection probabilities, but also, crucially, uncertainty estimates. We indicate how such results can then be used to obtain optimal conclusions about presence of bacteria, and illustrate how strongly the sampling results speak in favour of or against contamination. In our example, we consider the case when B. cereus is used as surrogate for B. anthracis, for safety reasons. The statistical modelling of the detection probabilities and of the growth characteristics of the bacteria types is based on data from four experiments where different matrices of food were spiked with B. anthracis or B. cereus and analysed using plate counts and qPCR. We show how flexible and complex Bayesian models, together with inference tools such as OpenBUGS, can be used to merge information about detection probability curves. Two different modelling approaches, differing in whether the pre-enrichment step and the PCR detection step are modelled separately or together, are applied. The relative importance on the detection curves for various existing data sets are evaluated and illustrated.

  19. Recent Developments in Ecological Economics

    DEFF Research Database (Denmark)

    Reader with published articles within the field of ecological economics, mostly from 1997 - 2007......Reader with published articles within the field of ecological economics, mostly from 1997 - 2007...

  20. Quantitative plant ecology

    DEFF Research Database (Denmark)

    Damgaard, Christian

    2014-01-01

    This e-book is written in the Wolfram' CDF format (download free CDF player from Wolfram.com) The objective of this e-book is to introduce the population ecological concepts for measuring and predicting the ecological success of plant species. This will be done by focusing on the measurement...... and statistical modelling of plant species abundance and the relevant ecological processes that control species abundance. The focus on statistical modelling and likelihood function based methods also means that more algorithm based methods, e.g. ordination techniques and boosted regression tress...

  1. Complex adaptive systems ecology

    DEFF Research Database (Denmark)

    Sommerlund, Julie

    2003-01-01

    In the following, I will analyze two articles called Complex Adaptive Systems EcologyI & II (Molin & Molin, 1997 & 2000). The CASE-articles are some of the more quirkyarticles that have come out of the Molecular Microbial Ecology Group - a groupwhere I am currently making observational studies....... They are the result of acooperation between Søren Molin, professor in the group, and his brother, JanMolin, professor at Department of Organization and Industrial Sociology atCopenhagen Business School. The cooperation arises from the recognition that bothmicrobial ecology and sociology/organization theory works...

  2. [Parasitism and ecological parasitology].

    Science.gov (United States)

    Balashov, Iu S

    2011-01-01

    Parasitism as one of the life modes is a general biological phenomenon and is a characteristic of all viruses, many taxa of bacteria, fungi, protists, metaphytes, and metazoans. Zooparasitology is focused on studies of parasitic animals, particularly, on their taxonomy, anatomy, life cycles, host-parasite relations, biocoenotic connections, and evolution. Ecological parasitology is a component of ecology, as the scientific study of the relation of living organisms with each other and their surroundings. In the present paper, critical analysis of the problems, main postulates, and terminology of the modern ecological parasitology is given.

  3. Progress on the Molecular Genotyping Techniques of Bacillus anthracis%炭疽芽孢杆菌分子分型研究进展

    Institute of Scientific and Technical Information of China (English)

    吴松羽; 刘先凯; 宋丽; 魏华; 王恒樑

    2011-01-01

    随着分子生物学的发展,分子分型技术被广泛应用于鉴别炭疽芽孢杆菌菌株间的遗传相关性和流行病学特征.我们就近年来常用的炭疽芽孢杆菌分子分型方法的优缺点和研究进展进行综述.%With the development of molecular biology, genotyping techniques are widely used in identification of strains of Bacillus anthracis in the genetic correlation and epidemiological characteristics. In this paper, we reviewed the advantages and disadvantages of the commonly methods used in genotyping of B.anthracis in recent years.

  4. Development of a killed but metabolically active anthracis vaccine candidate strain%一种KBMA炭疽疫苗候选株的研制

    Institute of Scientific and Technical Information of China (English)

    沈非; 刘纯杰; 袁盛凌; 展德文; 王艳春; 任敏; 陶好霞; 王芃; 王令春; 陈冬生

    2011-01-01

    Anthrax is a zoonosis caused by Bacillus anthracis, which seriously affects human health. In recent years, a special phenomenon is found that the metabolic active of a bacterium remains after it is killed. To development of a KBMA (killed but metabolically active) Bacillus anthracis vaccine candidate strain, a plasmid pMAD and a recombinase system Cre-loxP were used to knockout the uvrAB gene of B. anthracis AP422 which lacks both of two plasmids pXOl and pXO2. The results of PCR and RT-PCR shows that uvrAB genes were deleted from B. anthracis AP422 chromosome successfully. The constructed B. anthracis AP422ΔuvrAB was inactivated by photochemical treatment (PCT) including an exposure in a long-wave-length ultraviolet (UVA)light and a treatment of 8-Methoxypsoralen (8-MOP), then the metabolic activity were detected by the method of MTS. The results showed that the killed B. anthracis AP422ΔuvrAB maintained a highly metabolic activity for at least 4 hours, showing a state of KBMA. The KBMA strain of B. anthracis AP422ΔuvrAB provides the prospective vaccine candidate strain for anthrax.%炭疽病是由炭疽芽胞杆菌Bacillus anthracis 引起的一种人畜共患传染病,严重影响着人类的健康.近年来在细菌疫苗的研究中发现一种特殊的现象:细菌被杀死后,体内的代谢活性却仍然维持(Killed but metabolically active,KBMA).此发现为炭疽新型疫苗候选株的研制提供了新思路.先通过同源重组的方法,利用pMAD质粒和Cre-loxP重组酶系统完成对缺失两个毒性大质粒的炭疽芽胞杆菌减毒株AP422的uvrAB基因的敲除,得到AP422△uvrAB菌株,然后通过光化学处理(包括长波紫外光的照射和8-甲氧基补骨脂素处理),使炭疽芽胞杆菌AP422△uvrAB失去繁殖能力.利用四氮唑化合物MTS检测其代谢活性,表明光化学处理杀死后的炭疽芽胞杆菌AP422△uvrAB在至少4 h内维持一个很高的代谢活性水平,即具备

  5. Marine Ecological Environment Management Based on Ecological Compensation Mechanisms

    Directory of Open Access Journals (Sweden)

    Qunzhen Qu

    2016-12-01

    Full Text Available The level of marine environmental management is a key factor in the successful implementation of marine power strategies. The improvement in management levels of marine environments requires innovation in marine management. In other words, the transformation of marine environmental management into marine ecological environment management must be done in order to achieve sustainable development of the marine economy. As an environmental economic policy that combines both administrative and market measures, ecological compensation mechanisms have significant advantages in marine ecological environment management. Based on the study of the current development of ecological compensation mechanisms in China, through the analysis of the connotation of marine ecological civilization, existing marine ecological protection practices and marine environmental management methods, this paper posits that the current marine ecological environment management in China should be established on the basis of ecological compensation mechanisms. At present, a lack of laws and regulations for overall marine ecological environment management is the key factor restricting the practice of marine ecological environment management. Therefore, it is necessary to explore the current path of marine ecological environment management in China from the perspective of the construction of legal system of ecological compensation law, the establishment of ecological compensation fees, ecological taxes and ecological compensation fund systems, and the clear status for a marine ecological management and supervision body.

  6. Green Turtle Trophic Ecology

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — SWFSC is currently conducting a study of green sea turtle (Chelonia mydas) trophic ecology in the eastern Pacific. Tissue samples and stable carbon and stable...

  7. Market Squid Ecology Dataset

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This dataset contains ecological information collected on the major adult spawning and juvenile habitats of market squid off California and the US Pacific Northwest....

  8. Ecological Provinces of Minnesota

    Data.gov (United States)

    Minnesota Department of Natural Resources — This coverage provides information for the first level of the Ecological Classification System. The boundaries of the polygons of this coverage were derived from...

  9. Ecological Subsections of Minnesota

    Data.gov (United States)

    Minnesota Department of Natural Resources — This coverage provides information for the third level of the Ecological Classification System. The boundaries of the polygons of this coverage were derived from...

  10. Ecological Sections of Minnesota

    Data.gov (United States)

    Minnesota Department of Natural Resources — This coverage provides information for the second level of the Ecological Classification System. The boundaries of the polygons of this coverage were derived from...

  11. Characterization of Bacillus anthracis-Like Bacteria Isolated from Wild Great Apes from Côte d'Ivoire and Cameroon

    OpenAIRE

    Klee, Silke R.; Özel, Muhsin; Appel, Bernd; Boesch, Christophe; Ellerbrok, Heinz; Jacob, Daniela; Holland, Gudrun; Fabian H Leendertz; Pauli, Georg; Grunow, Roland; Nattermann, Herbert

    2006-01-01

    We present the microbiological and molecular characterization of bacteria isolated from four chimpanzees and one gorilla thought to have died of an anthrax-like disease in Côte d'Ivoire and Cameroon. These isolates differed significantly from classic Bacillus anthracis by the following criteria: motility, resistance to the gamma phage, and, for isolates from Cameroon, resistance to penicillin G. A capsule was expressed not only after induction by CO2 and bicarbonate but also under normal grow...

  12. Cloning and Expression of Fusion Genes of Domain A-1 Protective Antigen of Bacillus Anthracis and Shigella Enterotoxin B Subunit (Stxb In E. Coil

    Directory of Open Access Journals (Sweden)

    AH ahmadi

    2015-02-01

    Conclusion: The findings of the current study revealed that this antigen can be raised as an anti-cancer and recombinant vaccine candidate against types of Shigella, Escherichia coli and Bacillus anthracis which can be due to such factors as identification of antigen(PA by antibody PA20, its apoptosis induction properties, property of immunogenicity, adjuvant and delivery of STxB protein and high expression levels of Gb3 in human cancer cells.

  13. Two new sequence type isolates of Bacillus anthracis by multilocus sequence typing%两株炭疽芽胞杆菌MLST新序列型(ST)

    Institute of Scientific and Technical Information of China (English)

    左庭婷; 李岩伟; 韩雪莲; 何君; 端青

    2012-01-01

    [目的]2株炭疽芽胞杆菌(Bacillus anthracis)17003-14和17003-32的多位点序列分型(Multilocussequence typing,MLST)研究.[方法]选取B.anthracis基因组7个常见管家基因位点glpF、gmk、ilvD、pta、pur、pycA和tpi进行PCR扩增、测序,与MLST数据库中的等位基因序列进行比对,确定菌株的序列型( sequence type,ST).[结果]B.anthracis 17003-14和17003-32的等位基因编号分别为113、31、1、43、1、53、7和113、31、1、43、1、53、37,比对结果显示这2株细菌的等位基因编号组合未见报道.[结论]17003-14和17003-32为新ST菌株,已被MLST数据库确认,注册号(pubMLST id)分别为id-1053和id-1054.%[Objective] To define the sequence type (ST) isolates of Bacillus anthracis by multilocus sequence typing (MLST). [Methods] Fragments of seven housekeeping genes (glpF, gmk, ilvD, pta, pur, pycA , and tpi) were amplified by PCR using the standard primers as described on the website for MLST of Bacillus and the sequences were compared with existing allele sequences on the MLST website. [Results] Two novel allele combinations of the seven loci were found in two isolates 17003-14 and 17003-32. [Conclusion] Two novel ST isolates of B. anthracis were identified by this study and confirmed by the MLST website, and the pubMLST ids were id-1053 and id-1054.

  14. Matrix Assisted Laser Desorption Ionization Mass Spectrometric Analysis of Bacillus anthracis: From Fingerprint Analysis of the Bacterium to Quantification of its Toxins in Clinical Samples

    Science.gov (United States)

    Woolfitt, Adrian R.; Boyer, Anne E.; Quinn, Conrad P.; Hoffmaster, Alex R.; Kozel, Thomas R.; de, Barun K.; Gallegos, Maribel; Moura, Hercules; Pirkle, James L.; Barr, John R.

    A range of mass spectrometry-based techniques have been used to identify, characterize and differentiate Bacillus anthracis, both in culture for forensic applications and for diagnosis during infection. This range of techniques could usefully be considered to exist as a continuum, based on the degrees of specificity involved. We show two examples here, a whole-organism fingerprinting method and a high-specificity assay for one unique protein, anthrax lethal factor.

  15. GneZ, a UDP-GlcNAc 2-epimerase, is required for S-layer assembly and vegetative growth of Bacillus anthracis.

    Science.gov (United States)

    Wang, Ya-Ting; Missiakas, Dominique; Schneewind, Olaf

    2014-08-15

    Bacillus anthracis, the causative agent of anthrax, forms an S-layer atop its peptidoglycan envelope and displays S-layer proteins and Bacillus S-layer-associated (BSL) proteins with specific functions to support cell separation of vegetative bacilli and growth in infected mammalian hosts. S-layer and BSL proteins bind via the S-layer homology (SLH) domain to the pyruvylated secondary cell wall polysaccharide (SCWP) with the repeat structure [→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→]n, where α-GlcNAc and β-GlcNAc are substituted with two and one galactosyl residues, respectively. B. anthracis gneY (BAS5048) and gneZ (BAS5117) encode nearly identical UDP-GlcNAc 2-epimerase enzymes that catalyze the reversible conversion of UDP-GlcNAc and UDP-ManNAc. UDP-GlcNAc 2-epimerase enzymes have been shown to be required for the attachment of the phage lysin PlyG with the bacterial envelope and for bacterial growth. Here, we asked whether gneY and gneZ are required for the synthesis of the pyruvylated SCWP and for S-layer assembly. We show that gneZ, but not gneY, is required for B. anthracis vegetative growth, rod cell shape, S-layer assembly, and synthesis of pyruvylated SCWP. Nevertheless, inducible expression of gneY alleviated all the defects associated with the gneZ mutant. In contrast to vegetative growth, neither germination of B. anthracis spores nor the formation of spores in mother cells required UDP-GlcNAc 2-epimerase activity.

  16. Sraffa and ecological economics

    OpenAIRE

    Verger, Yoann

    2015-01-01

    References to Sraffa and to the neo-Ricardian school is something quite customary in ecological economics. By looking at contributions in this area since the beginning of ecological economics and at contributions on environmental problem from the neo-Ricardian school, we see that a connection between both school still has to be made. This connection should be articulated around the initial aim of Sraffa: to develop a new paradigm, competing against the neoclassical one. Only then it will be p...

  17. Indicators of Ecological Change

    Science.gov (United States)

    2005-03-03

    Wang et al. (2003) and earlier those of Chapra and Di Toro (1991). Wang et al. (2003) termed their approach the extreme value method and used it to...Thesis, Auburn University, Auburn, AL. 210 pp. Chapra , S. C., Di Toro, D. M., 1991. Delta method for estimating primary production, respiration, and...characteristics, The challenge in using ecological indicators is determining which of the numerous measures of ecological systems best characterize

  18. Urban Sound Ecologies

    DEFF Research Database (Denmark)

    Groth, Sanne Krogh; Samson, Kristine

    2013-01-01

    of the 1970s while developing them into recent definitions of ecology in urban studies. Finally, we unfold our framing of urban sound ecologies with three case analyses: a sound intervention in Berlin, a symphony for wind instruments in Copenhagen and a video walk in a former railway station in Kassel...... to how artists working with new information and media technologies create inventive ways of inserting sound and image into urban environments....

  19. Ecological Perspectives in HCI

    DEFF Research Database (Denmark)

    Blevis, Eli; Bødker, Susanne; Flach, John;

    The aim of the workshop is to provide a forum for researchers and practitioners to discuss the present and future of ecological perspectives in HCI. The participants will reflect on the current uses and interpretations of “ecology” and related concepts in the field. The workshop will assess...... the potential of ecological perspectives in HCI for supporting rich and meaningful analysis, as well as innovative design, of interactive technologies in real-life contexts...

  20. Developments in numerical ecology

    Energy Technology Data Exchange (ETDEWEB)

    Legendre, P.; Legendre, L.

    1987-01-01

    From earlier ecological studies it has become apparent that simple univariate or bivariate statistics are often inappropriate, and that multivariate statistical analyses must be applied. Despite several difficulties arising from the application of multivariate methods, community ecology has acquired a mathematical framework, with three consequences: it can develop as an exact science; it can be applied operationally as a computer-assisted science to the solution of environmental problems; and it can exchange information with other disciplines using the language of mathematics.

  1. A survey of the occurrence of Bacillus anthracis in North American soils over two long-range transects and within post-Katrina New Orleans

    Science.gov (United States)

    Griffin, Dale W.; Petrosky, Terry; Morman, Suzette A.; Luna, Vicki A.

    2009-01-01

    Soil samples were collected along a north-south transect extending from Manitoba, Canada, to the US-Mexico border near El Paso, Texas in 2004 (104 samples), a group of sites within New Orleans, Louisiana following Hurricane Katrina in 2005 (19 samples), and a Gulf Coast transect extending from Sulphur, Louisiana, to DeFuniak Springs, Florida, in 2007 (38 samples). Samples were collected from the top 40 cm of soil and were screened for the presence of total Bacillus species and Bacillus anthracis (anthrax), specifically using multiplex-polymerase chain reaction (PCR). Using an assay with a sensitivity of ~170 equivalent colony-forming units (CFU) g-1 field moist soil, the prevalence rate of Bacillus sp./B. anthracis in the north-south transect and the 2005 New Orleans post-Katrina sample set were 20/5% and 26/26%, respectively. Prevalence in the 2007 Gulf Coast sample set using an assay with a sensitivity of ~4 CFU g-1 of soil was 63/0%. Individual transect-set data indicate a positive relation between occurrences of species and soil moisture or soil constituents (i.e., Zn and Cu content). The 2005 New Orleans post-Katrina data indicated that B. anthracis is readily detectable in Gulf Coast soils following flood events. The data also indicated that occurrence, as it relates to soil chemistry, may be confounded by flood-induced dissemination of germinated cells and the mixing of soil constituents for short temporal periods following an event.

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

    Directory of Open Access Journals (Sweden)

    Shawn G. Gibbs

    2014-05-01

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

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

    Science.gov (United States)

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

    2016-01-06

    Anthrax is a lethal disease caused by the gram-positive spore-producing bacterium Bacillus anthracis. Live attenuated vaccines, such as the nonencapsulated Sterne strain, do not meet the safety standards mandated for human use in the Western world and are approved for veterinary purposes only. Here we demonstrate that disrupting the htrA gene, encoding the chaperone/protease HtrA (High Temperature Requirement A), in the virulent Bacillus anthracis Vollum strain results in significant virulence attenuation in guinea pigs, rabbits and mice, underlying the universality of the attenuated phenotype associated with htrA knockout. Accordingly, htrA disruption was implemented for the development of a Sterne-derived safe live vaccine compatible with human use. The novel B. anthracis SterneΔhtrA strain secretes functional anthrax toxins but is 10-10(4)-fold less virulent than the Sterne vaccine strain depending on animal model (mice, guinea pigs, or rabbits). In spite of this attenuation, double or even single immunization with SterneΔhtrA spores elicits immune responses which target toxaemia and bacteremia resulting in protection from subcutaneous or respiratory lethal challenge with a virulent strain in guinea pigs and rabbits. The efficacy of the immune-protective response in guinea pigs was maintained for at least 50 weeks after a single immunization.

  4. The effect of prolonged storage on the virulence of isolates of Bacillus anthracis obtained from environmental and animal sources in the Kars Region of Turkey.

    Science.gov (United States)

    Buyuk, Fatih; Sahin, Mitat; Cooper, Callum; Celebi, Ozgur; Saglam, Aliye Gulmez; Baillie, Les; Celik, Elif; Akca, Dogan; Otlu, Salih

    2015-07-01

    The stability of the plasmid-mediated virulence factors of Bacillus anthracis, a tripartite toxin located on pXO1 and an antiphagocytic capsule encoded by genes located on pXO2, following long-term storage was investigated. A collection of 159 isolates of B. anthracis were collected from the Kars region of Turkey between 2000 and 2013 and stored at -20°C in Brucella broth supplemented with 20% glycerine. A total of 142 isolates were recovered of which one failed to express a capsule upon primary culture. A further 35 isolates yielded a mixture of mucoid and non-mucoid colonies; the majority of which had lost the pXO2 plasmid as determined by PCR analysis. Results would suggest that pXO2 is more unstable than pXO1 and that this instability increases with the length of storage. It is possible that the pXO2-deficient isolates of B. anthracis described here could be developed into a vaccine to treat at risk animals in the Kars region as many animal vaccines are based upon pXO2 deficiency.

  5. Molecular diversity of Bacillus anthracis in the Netherlands: investigating the relationship to the worldwide population using whole-genome SNP discovery.

    Science.gov (United States)

    Derzelle, Sylviane; Girault, Guillaume; Roest, Hendrik Ido Jan; Koene, Miriam

    2015-06-01

    Bacillus anthracis, the causative agent of anthrax, has been widely described as a clonal species. Here we report the use of both canonical SNP analysis and whole-genome sequencing to characterize the phylogenetic lineages of B. anthracis from the Netherlands. Eleven strains isolated over a 25-years period (1968-1993) were paired-end sequenced using parallel sequencing technology. Five canSNP groups or lineages, i.e. A.Br.001/002 (n=6), A.Br.Aust94 (n=2), A.Br.008/011 (n=1), A.Br.011/009 (n=1) and A.Br.Vollum (n=1) were identified. Comparative analyses, with a focus on SNPs discovery, were carried out using a total of 52 B. anthracis genomes. A phylogeographic "Dutch" cluster within the dominant A.Br.001/002 group was discovered, involving isolates from a single outbreak. Diagnostic SNPs specific to the newly identified sub-groups were developed into high-resolution melting SNP discriminative assays for the purpose of rapid molecular epidemiology. Phylogenetic relationships with strains from other parts of the world are discussed.

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

    Science.gov (United States)

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

    2016-01-01

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

  7. 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.

  8. Benefits of ecological engineering practices

    NARCIS (Netherlands)

    Brüll, A.; Van Bohemen, H.; Costanza, R.; Mitsch, W.J.

    2011-01-01

    With the intention to further promote the field of ecological engineering and the solutions it provides, a workshop on “Benefits of Ecological Engineering Practices” was held 3 Dec 2009. It was conducted by the International Ecological Engineering Society in Paris at the conference “Ecological Engin

  9. Urban ecology and its Importance

    Institute of Scientific and Technical Information of China (English)

    艾玛诺

    2015-01-01

    With the development of ecological problems are becoming more and more serious people pay more and more attention on ecological city,, this paper analyzes the sources of city ecological problems from the angle of the city planning, to explore the feasible measures to solve or al eviate the increasingly serious ecological problems.

  10. Benefits of ecological engineering practices

    OpenAIRE

    Brüll, A.; Van Bohemen, H.; Costanza, R.; Mitsch, W.J.

    2011-01-01

    With the intention to further promote the field of ecological engineering and the solutions it provides, a workshop on “Benefits of Ecological Engineering Practices” was held 3 Dec 2009. It was conducted by the International Ecological Engineering Society in Paris at the conference “Ecological Engineering: from Concepts to Application” organized by the Ecological Engineering Applications Group GAIE. This paper presents the results of the workshop related to three key questions: (1) what are t...

  11. Urban sound ecologies

    Directory of Open Access Journals (Sweden)

    Sanne Krogh Groth

    2013-12-01

    Full Text Available Within recent years, there has been a renewed focus on sound in urban environments. From sound installations in public space to sound festivals in alternative settings, we find a common interest in sound art relating to the urban environment. Artworks or interventions presented in such contexts share the characteristics of site specificity. However, this article will consider the artwork in a broader context by re-examining how sound installations relate to the urban environment. For that purpose, this article brings together ecology terms from acoustic ecology of the sound theories of the 1970s while developing them into recent definitions of ecology in urban studies. Finally, we unfold our framing of urban sound ecologies with three case analyses: a sound intervention in Berlin, a symphony for wind instruments in Copenhagen and a video walk in a former railway station in Kassel. The article concludes that the ways in which recent sound installations work with urban ecologies vary. While two of the examples blend into the urban environment, the other transfers the concert format and its mode of listening to urban space. Last, and in accordance with recent soundscape research, we point to how artists working with new information and media technologies create inventive ways of inserting sound and image into urban environments.

  12. ECOLOGICAL ECONOMICS VS ECONOMIC(AL ECOLOGY

    Directory of Open Access Journals (Sweden)

    G. Kharlamova

    2015-10-01

    Full Text Available Currently world faces the dilemma – ecological economy or economic(al ecology. The researchers produce hundreds of surveys on the topic. However the analyses of recent most cited simulations had shown the diversity of results. Thus, for some states the Kuznets environmental curve has place, for others – no. Same could be said about different years for the same state. It provokes the necessity of drawing new group analyses to reveal the tendencies and relationships between economic and environmental factors. Most flexible and mirror factor of environmental sustainability is the volume of CO2 emissions. The econometric analysis was used for detecting the economic impact on this indicator at the global level and in the spectra of group of states depending on their income. The hypothesis of the existence of environmental Kuznets curve for the analysed data is rejected. Real GDP per capita impact on carbon dioxide emissions is considered only at the global level. The impact of openness of the economy is weak. Rejection happened also to the hypothesis that for the developed countries there is a reverse dependence between the environmental pollution and economic openness. Indicator “energy consumption per capita” impacts on greenhouse gas emissions only in countries with high income. Whereby it should be noted that the more developed a country is, the more elastic is this influence. These results have a potential usage for environmental policy regulation and climate strategy.

  13. Ecological city planning

    Directory of Open Access Journals (Sweden)

    Salvador Rueda

    2013-07-01

    Full Text Available A territory, a city, a neighbourhood are all ecosystems; a mixture of chemico-physical and organic elements related to each other. That which defines an ecological system is the set of rules and characteristics which condition its relationships, and its duration in time is guaranteed by its efficiency and internal organization which applied to the city is translated in the reduction of the use of natural resources and in the increase of social organization. To increase the efficiency of the urban systems is the necessary condition for the formulation of ecological city planning favouring the maximum liveability of sites. Liveability is directly correlated to the optimization of numerous elements (public space, equipment, services, building techniques, innovative technology, social cohesion, biodiversity. To carry out such objectives, ecological city planning proposes a new model of town planning on three levels (subsoil, ground level, and upper level.

  14. Coevolutionary ecological economics

    Energy Technology Data Exchange (ETDEWEB)

    Kallis, Giorgos [ICREA Researcher, ICTA, Universidad Autonoma de Barcelona, ETSE, QC/3095, 08193 Bellatera, Barcelona (Spain); Norgaard, Richard B. [Energy and Resources Group, University of California at Berkeley, 310 Barrows Hall, Berkeley, CA, 94720-3050 (United States)

    2010-02-15

    This paper maps a coevolutionary research agenda for ecological economics. At an epistemological level coevolution offers a powerful logic for transcending environmental and social determinisms and developing a cross-disciplinary approach in the study of socio-ecological systems. We identify four consistent stories emerging out of coevolutionary studies in ecological economics, concerning: environmental degradation and development failure in peripheral regions; the lock-in of unsustainable production-consumption patterns; the vicious cycle between human efforts to control undesirable micro-organisms and the evolution of these organisms; and the adaptive advantages of other-regarding, cooperative behaviors and institutions. We identify challenges in the conceptualization of coevolutionary relationships in relation to: the interaction between different hierarchical levels of evolution; the role of space and social power; uneven rates of change and crises. We conclude with the political implications of a coevolutionary perspective based on the premises of pragmatism. (author)

  15. Ecology of gelatious plankton

    DEFF Research Database (Denmark)

    Jaspers, Cornelia

    as a result of this invasion and its ecological and economic impacts. In 2005, when M. leidyi was sighted in Northern Europe for the first time, similar consequences were feared. The aim of my PhD project was to understand the potential impact of M. leidyi on the Baltic Sea ecosystem and constrains on its...... in high and intermediate saline areas in Northern Europe. While the ecological impact of M. leidyi in the central Baltic appears to be limited concern, the environment in other European waters should be more favourable to their populations. In these areas, it is suggested that M. leidyi constitutes...

  16. Art, Ecology and Institutions

    DEFF Research Database (Denmark)

    Witzke, Anne Sophie

    2013-01-01

    The discourse of ecology and sustainability has gained critical traction in recent years. But how are these concepts framed within the space, language and idea of the exhibition? This panel discussion, moderated by Steven Lam and conducted by email in July 2012, sought to unpack the claims...... and limits of the ecological, looking specifically at various international case studies, within the practice of curatorial and exhibition studies. The discussion begins with a reflection on ‘DON'T/PANIC’ in Durban and ‘Rethink – Contemporary Art and Climate Change’ in Copenhagen, exhibitions that were...

  17. 炭疽芽胞杆菌(Bacillus anthracis)检测质粒的构建及其应用%Construction of Plasmid pBIB2006 for Detecting Bacillus anthracis and Its Application

    Institute of Scientific and Technical Information of China (English)

    吕和平; 闵勇; 杨东明; 郑应华; 李旭; 武轶

    2007-01-01

    根据炭疽芽胞杆菌(Bacillus anthracis)毒性质粒pX01和pX02上的2个毒力相关基因cya和capA的序列特点,以pIJ2925为出发载体,采用一步重叠延伸PCR技术(One-step Overlap Extension PCR,简称OOE-PCR)构建了包含cya基因和capA基因保守区DNA片段的炭疽检测质粒pBIB2006.采用复合PCR对模拟炭疽危险品进行分析,结果表明pBIB2006可以为炭疽芽胞杆菌的检测提供准确、安全和方便的阳性参照品,从而为检测炭疽芽胞杆菌和炭疽芽胞杆菌灭活疫苗提供了便利.

  18. The development of ecological tourism and ecological civilization construction

    Institute of Scientific and Technical Information of China (English)

    张波

    2014-01-01

    This passage shows us the characters of ecology tourism and reveals the problems in the modern tourism , emphasises that we should develop ecology tourism and make a link between them, then leads to Sustainable Development.

  19. Biodiversity in Benthic Ecology

    DEFF Research Database (Denmark)

    Friberg, Nikolai; Carl, J. D.

    Foreword: This proceeding is based on a set of papers presented at the second Nordic Benthological Meeting held in Silkeborg, November 13-14, 1997. The main theme of the meeting was biodiversity in benthic ecology and the majority of contributions touch on this subject. In addition, the proceeding...

  20. Activity Book: Ocean Ecology.

    Science.gov (United States)

    Learning, 1992

    1992-01-01

    Presents a collection of activities to help elementary students study ocean ecology. The activities have students investigate ocean inhabitants, analyze animal adaptations, examine how temperature and saltiness affect ocean creatures, and learn about safeguarding the sea. Student pages offer reproducible learning sheets. (SM)

  1. Ecology and Human Destiny.

    Science.gov (United States)

    Haught, John F.

    1999-01-01

    Examines eschatology as the heart of Christian faith, suggesting that an appreciation of an eschatological interpretation of the cosmos enables acceptance of nature's transience and a grounding for an ecological ethic. Maintains that recent scientific developments present a promising, rather than pessimistic, picture of the universe. Holds that…

  2. Ecology in Urban Education.

    Science.gov (United States)

    Johnson, Letitia K.; Ryan, Michael

    In this course guide to the teaching of urban ecology, six learning activities on the following topics are outlined: (1) city location and growth; (2) an in-depth study of New Orleans; (3) city shape and structure; (4) size and spacing of cities; (5) cities with special functions; (6) local community study. Educational objectives for each activity…

  3. Challenges of ecological restoration

    DEFF Research Database (Denmark)

    Halme, Panu; Allen, Katherine A.; Aunins, Ainars

    2013-01-01

    The alarming rate of ecosystem degradation has raised the need for ecological restoration throughout different biomes and continents. North European forests may appear as one of the least vulnerable ecosystems from a global perspective, since forest cover is not rapidly decreasing and many ecosys...

  4. Metabolomics in chemical ecology.

    Science.gov (United States)

    Kuhlisch, Constanze; Pohnert, Georg

    2015-07-01

    Chemical ecology elucidates the nature and role of natural products as mediators of organismal interactions. The emerging techniques that can be summarized under the concept of metabolomics provide new opportunities to study such environmentally relevant signaling molecules. Especially comparative tools in metabolomics enable the identification of compounds that are regulated during interaction situations and that might play a role as e.g. pheromones, allelochemicals or in induced and activated defenses. This approach helps overcoming limitations of traditional bioassay-guided structure elucidation approaches. But the power of metabolomics is not limited to the comparison of metabolic profiles of interacting partners. Especially the link to other -omics techniques helps to unravel not only the compounds in question but the entire biosynthetic and genetic re-wiring, required for an ecological response. This review comprehensively highlights successful applications of metabolomics in chemical ecology and discusses existing limitations of these novel techniques. It focuses on recent developments in comparative metabolomics and discusses the use of metabolomics in the systems biology of organismal interactions. It also outlines the potential of large metabolomics initiatives for model organisms in the field of chemical ecology.

  5. THE AUTOIMMUNE ECOLOGY.

    Directory of Open Access Journals (Sweden)

    Juan-Manuel eAnaya

    2016-04-01

    Full Text Available Autoimmune diseases (ADs represent a heterogeneous group of disorders that affect specific target organs or multiple organ systems. These conditions share common immunopathogenic mechanisms (i.e., the autoimmune tautology, which explain the clinical similarities they have among them as well as their familial clustering (i.e., coaggregation. As part of the autoimmune tautology, the influence of environmental exposure on the risk of developing ADs is paramount (i.e., the autoimmune ecology. In fact, environment, more than genetics, shapes immune system. Autoimmune ecology is akin to exposome, that is all the exposures - internal and external - across the lifespan, interacting with hereditary factors (both genetics and epigenetics to favor or protect against autoimmunity and its outcomes. Herein we provide an overview of the autoimmune ecology, focusing on the immune response to environmental agents in general, and microbiota, cigarette smoking, alcohol and coffee consumption, socioeconomic status, gender and sex hormones, vitamin D, organic solvents and vaccines in particular. Inclusion of the autoimmune ecology in disease etiology and health will improve the way personalized medicine is currently conceived and applied.

  6. Ecology of lianas

    NARCIS (Netherlands)

    Schnitzer, S.A.; Bongers, F.; Burnham, R.J.; Putz, F.E.

    2015-01-01

    A liana is a long-stemmed, woody vine that is rooted in the soil at ground level and uses trees to climb up to the canopy to get access to well-lit areas of the forest. The main goal of this book is to present the current status of liana ecology in tropical and temperate forests. In essence, it is a

  7. Aspects of Marine Ecology.

    Science.gov (United States)

    Awkerman, Gary L.

    This publication is designed for use in standard science curricula to develop oceanologic manifestations of certain science topics. Included are teacher guides, student activities, and demonstrations to impart ocean science understanding, specifically, aspects of marine ecology, to high school students. The course objectives include the ability of…

  8. Marine and Island Ecology.

    Science.gov (United States)

    Stephens, Lawrence J.; And Others

    1988-01-01

    Describes an ecology course which provides students with an opportunity to observe aquatic and terrestrial life in the Bahamas. States that students learn scientific methodology by measuring physical and chemical aspects of the island habitats. Provides information on the island, course description and objectives, transportation, facilities, and…

  9. The Autoimmune Ecology.

    Science.gov (United States)

    Anaya, Juan-Manuel; Ramirez-Santana, Carolina; Alzate, Maria A; Molano-Gonzalez, Nicolas; Rojas-Villarraga, Adriana

    2016-01-01

    Autoimmune diseases (ADs) represent a heterogeneous group of disorders that affect specific target organs or multiple organ systems. These conditions share common immunopathogenic mechanisms (i.e., the autoimmune tautology), which explain the clinical similarities they have among them as well as their familial clustering (i.e., coaggregation). As part of the autoimmune tautology, the influence of environmental exposure on the risk of developing ADs is paramount (i.e., the autoimmune ecology). In fact, environment, more than genetics, shapes immune system. Autoimmune ecology is akin to exposome, that is all the exposures - internal and external - across the lifespan, interacting with hereditary factors (both genetics and epigenetics) to favor or protect against autoimmunity and its outcomes. Herein, we provide an overview of the autoimmune ecology, focusing on the immune response to environmental agents in general, and microbiota, cigarette smoking, alcohol and coffee consumption, socioeconomic status (SES), gender and sex hormones, vitamin D, organic solvents, and vaccines in particular. Inclusion of the autoimmune ecology in disease etiology and health will improve the way personalized medicine is currently conceived and applied.

  10. gyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group

    Science.gov (United States)

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

    2004-01-01

    Bacillus anthracis, the causative agent of the human disease anthrax, Bacillus cereus, a food-borne pathogen capable of causing human illness, and Bacillus thuringiensis, a well-characterized insecticidal toxin producer, all cluster together within a very tight clade (B. cereus group) phylogenetically and are indistinguishable from one another via 16S rDNA sequence analysis. As new pathogens are continually emerging, it is imperative to devise a system capable of rapidly and accurately differentiating closely related, yet phenotypically distinct species. Although the gyrB gene has proven useful in discriminating closely related species, its sequence analysis has not yet been validated by DNA:DNA hybridization, the taxonomically accepted "gold standard". We phylogenetically characterized the gyrB sequences of various species and serotypes encompassed in the "B. cereus group," including lab strains and environmental isolates. Results were compared to those obtained from analyses of phenotypic characteristics, 16S rDNA sequence, DNA:DNA hybridization, and virulence factors. The gyrB gene proved more highly differential than 16S, while, at the same time, as analytical as costly and laborious DNA:DNA hybridization techniques in differentiating species within the B. cereus group.

  11. Binding domains of Bacillus anthracis phage endolysins recognize cell culture age-related features on the bacterial surface.

    Science.gov (United States)

    Paskaleva, Elena E; Mundra, Ruchir V; Mehta, Krunal K; Pangule, Ravindra C; Wu, Xia; Glatfelter, Willing S; Chen, Zijing; Dordick, Jonathan S; Kane, Ravi S

    2015-01-01

    Bacteriolytic enzymes often possess a C-terminal binding domain that recognizes specific motifs on the bacterial surface and a catalytic domain that cleaves covalent linkages within the cell wall peptidoglycan. PlyPH, one such lytic enzyme of bacteriophage origin, has been reported to be highly effective against Bacillus anthracis, and can kill up to 99.99% of the viable bacteria. The bactericidal activity of this enzyme, however, appears to be strongly dependent on the age of the bacterial culture. Although highly bactericidal against cells in the early exponential phase, the enzyme is substantially less effective against stationary phase cells, thus limiting its application in real-world settings. We hypothesized that the binding domain of PlyPH may differ in affinity to cells in different Bacillus growth stages and may be primarily responsible for the age-restricted activity. We therefore employed an in silico approach to identify phage lysins differing in their specificity for the bacterial cell wall. Specifically we focused our attention on Plyβ, an enzyme with improved cell wall-binding ability and age-independent bactericidal activity. Although PlyPH and Plyβ have dissimilar binding domains, their catalytic domains are highly homologous. We characterized the biocatalytic mechanism of Plyβ by identifying the specific bonds cleaved within the cell wall peptidoglycan. Our results provide an example of the diversity of phage endolysins and the opportunity for these biocatalysts to be used for broad-based protection from bacterial pathogens.

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

    Institute of Scientific and Technical Information of China (English)

    Xiuli Dong; Eric McCoy; Mei Zhang; Liju Yang

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-12-01

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

  14. Monitoring biothreat agents (Francisella tularensis, Bacillus anthracis and Yersinia pestis) with a portable real-time PCR instrument.

    Science.gov (United States)

    Mölsä, Markos; Hemmilä, Heidi; Katz, Anna; Niemimaa, Jukka; Forbes, Kristian M; Huitu, Otso; Stuart, Peter; Henttonen, Heikki; Nikkari, Simo

    2015-08-01

    In the event of suspected releases or natural outbreaks of contagious pathogens, rapid identification of the infectious agent is essential for appropriate medical intervention and disease containment. The purpose of this study was to compare the performance of a novel portable real-time PCR thermocycler, PikoReal™, to the standard real-time PCR thermocycler, Applied Biosystems® 7300 (ABI 7300), for the detection of three high-risk biothreat bacterial pathogens: Francisella tularensis, Bacillus anthracis and Yersinia pestis. In addition, a novel confirmatory real-time PCR assay for the detection of F. tularensis is presented and validated. The results show that sensitivity of the assays, based on a dilution series, for the three infectious agents ranged from 1 to 100 fg of target DNA with both instruments. No cross-reactivity was revealed in specificity testing. Duration of the assays with the PikoReal and ABI 7300 systems were 50 and 100 min, respectively. In field testing for F. tularensis, results were obtained with the PikoReal system in 95 min, as the pre-PCR preparation, including DNA extraction, required an additional 45 min. We conclude that the PikoReal system enables highly sensitive and rapid on-site detection of biothreat agents under field conditions, and may be a more efficient alternative to conventional diagnostic methods.

  15. Molecular ecological network analyses

    Directory of Open Access Journals (Sweden)

    Deng Ye

    2012-05-01

    Full Text Available Abstract Background Understanding the interaction among different species within a community and their responses to environmental changes is a central goal in ecology. However, defining the network structure in a microbial community is very challenging due to their extremely high diversity and as-yet uncultivated status. Although recent advance of metagenomic technologies, such as high throughout sequencing and functional gene arrays, provide revolutionary tools for analyzing microbial community structure, it is still difficult to examine network interactions in a microbial community based on high-throughput metagenomics data. Results Here, we describe a novel mathematical and bioinformatics framework to construct ecological association networks named molecular ecological networks (MENs through Random Matrix Theory (RMT-based methods. Compared to other network construction methods, this approach is remarkable in that the network is automatically defined and robust to noise, thus providing excellent solutions to several common issues associated with high-throughput metagenomics data. We applied it to determine the network structure of microbial communities subjected to long-term experimental warming based on pyrosequencing data of 16 S rRNA genes. We showed that the constructed MENs under both warming and unwarming conditions exhibited topological features of scale free, small world and modularity, which were consistent with previously described molecular ecological networks. Eigengene analysis indicated that the eigengenes represented the module profiles relatively well. In consistency with many other studies, several major environmental traits including temperature and soil pH were found to be important in determining network interactions in the microbial communities examined. To facilitate its application by the scientific community, all these methods and statistical tools have been integrated into a comprehensive Molecular Ecological

  16. Valuation of ecological resources

    Energy Technology Data Exchange (ETDEWEB)

    Scott, M.J.; Bilyard, G.R.; Link, S.O.; Ricci, P.F.; Seely, H.E.; Ulibarri, C.A.; Westerdahl, H.E.

    1995-04-01

    Ecological resources are resources that have functional value to ecosystems. Frequently, these functions are overlooked in terms of the value they provide to humans. Environmental economics is in search of an appropriate analysis framework for such resources. In such a framework, it is essential to distinguish between two related subsets of information: (1) ecological processes that have intrinsic value to natural ecosystems; and (2) ecological functions that are values by humans. The present study addresses these concerns by identifying a habitat that is being displaced by development, and by measuring the human and ecological values associated with the ecological resources in that habitat. It is also essential to determine which functions are mutually exclusive and which are, in effect, complementary or products of joint production. The authors apply several resource valuation tools, including contingent valuation methodology (CVM), travel cost methodology (TCM), and hedonic damage-pricing (HDP). One way to derive upper-limit values for more difficult-to-value functions is through the use of human analogs, because human-engineered systems are relatively inefficient at supplying the desired services when compared with natural systems. Where data on the relative efficiencies of natural systems and human analogs exist, it is possible to adjust the costs of providing the human analog by the relative efficiency of the natural system to obtain a more realistic value of the function under consideration. The authors demonstrate this approach in an environmental economic case study of the environmental services rendered by shrub-steppe habitats of Benton County, Washington State.

  17. Ecological zones of California deserts

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — The dataset delineates ecological zones within California deserts. We derived ecological zones by reclassifying LANDFIRE vegetation biophysical setting types, plus...

  18. The redoubtable ecological periodic table

    Science.gov (United States)

    Ecological periodic tables are repositories of reliable information on quantitative, predictably recurring (periodic) habitat–community patterns and their uncertainty, scaling and transferability. Their reliability derives from their grounding in sound ecological principle...

  19. Genetic Evidence for the Involvement of the S-Layer Protein Gene sap and the Sporulation Genes spo0A, spo0B, and spo0F in Phage AP50c Infection of Bacillus anthracis

    OpenAIRE

    Roger D Plaut; Beaber, John W.; Zemansky, Jason; Kaur, Ajinder P.; George, Matroner; Biswas, Biswajit; Henry, Matthew; Bishop-Lilly, Kimberly A.; Mokashi, Vishwesh; Hannah, Ryan M.; Pope, Robert K.; Timothy D. Read; Stibitz, Scott; Calendar, Richard; Sozhamannan, Shanmuga

    2014-01-01

    In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles assoc...

  20. The Social-Ecological Ideal.

    Science.gov (United States)

    Gaidamak, A.; Tiittanen, T.

    1992-01-01

    Argues that it is essential for preschool education to explore environmental and ecological values. Discusses cognitive development of socio-ecological knowledge at three age levels. Asserts that folk tales provide good examples of ecological values because beauty usually triumphs over ugliness and good over evil. (CFR)

  1. Hanford Site Ecological Quality Profile

    Energy Technology Data Exchange (ETDEWEB)

    Bilyard, Gordon R.; Sackschewsky, Michael R.; Tzemos, Spyridon

    2002-02-17

    This report reviews the ecological quality profile methodology and results for the Hanford Site. It covers critical ecological assets and terrestrial resources, those in Columbia River corridor and those threatened and engdangered, as well as hazards and risks to terrestrial resources. The features of a base habitat value profile are explained, as are hazard and ecological quality profiles.

  2. Nutrition, ecology and nutritional ecology: towardan integrated framework

    DEFF Research Database (Denmark)

    Raubenheimer, David; Simpson, Steven J.; Mayntz, David

    2009-01-01

    1. The science of nutritional ecology spans a wide range of fields, including ecology, nutrition, behaviour, morphology, physiology, life history and evolutionary biology. But does nutritional ecology have a unique theoretical framework and research program and thus qualify as a field of research...... in its own right? 2. We suggest that the distinctive feature of nutritional ecology is its integrative nature, and that the field would benefit from more attention to formalizing a theoretical and quantitative framework for developing this. 3. Such a framework, we propose, should satisfy three minimal...... requirements: it should be nutritionally explicit, organismally explicit, and ecologically explicit. 4. We evaluate against these criteria four existing frameworks (Optimal Foraging Theory, Classical Insect Nutritional Ecology, the Geometric Framework for nutrition, and Ecological Stoichiometry), and conclude...

  3. 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.

  4. Establishment of Bacillus anthracis infection and its mechanism%炭疽芽孢杆菌建立感染的过程及其机制

    Institute of Scientific and Technical Information of China (English)

    刘炬; 徐俊杰; 陈薇

    2014-01-01

    Anthrax is a fulminating infectious disease .Bacillus anthracis, as the pathogen of anthrax , is a potential ma-terial for biological warfare agents and biological terrors .Immunoprophylaxis and specific theraphies play key roles in cop-ing with anthrax threats.Researches on the mechanism of B.anthracis infection, especially the process of infection , can fa-cilitate the development of novel drugs for anthrax prevention and therapy .Herein, by reviewing research progress , the in-fection process of B.anthracis is introduced and the potential mechanism of anthrax infection is described .Furthermore, the relationship between researches on anthrax infection mechanisms and the development of drugs for anthrax prevention and therapy is also discussed .%炭疽是一类烈性传染病,其病原体炭疽芽孢杆菌(简称炭疽菌)形成的芽孢是生物战剂和生物恐怖的原材料。免疫预防和特异性的治疗药物是应对这类生物威胁的重要手段。炭疽菌感染的机制研究,尤其是感染建立过程的研究能够为新型炭疽防治药物的研发提供新的思路。该文通过综述相关研究进展,介绍了炭疽菌的感染过程,描述了可能的感染机制,并结合炭疽防治药物的研究工作展开了讨论。

  5. Pyridine Nucleotide Complexes with Bacillus anthracis Coenzyme A-Disulfide Reductase: A Structural Analysis of Dual NAD(P)H Specificity

    Energy Technology Data Exchange (ETDEWEB)

    Wallen,J.; Paige, C.; Mallett, T.; Karplus, P.; Claiborne, A.

    2008-01-01

    We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis, and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 Angstroms resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides. The structures of the NADH and NADPH complexes at ca. 2.3 Angstroms resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367, 425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.

  6. Genotyping of French Bacillus anthracis strains based on 31-loci multi locus VNTR analysis: epidemiology, marker evaluation, and update of the internet genotype database.

    Directory of Open Access Journals (Sweden)

    Simon Thierry

    Full Text Available BACKGROUND: Bacillus anthracis is known to have low genetic variability. In spite of this lack of diversity, multiple-locus variable-number tandem repeat (VNTR analysis (MLVA and single nucleotide polymorphisms (SNPs including the canonical SNPs assay (canSNP have proved to be highly effective to differentiate strains. Five different MLVA schemes based on a collection of 31 VNTR loci (MLVA8, MLVA15, MLVA20, MLVA25 and MLVA31 with increased resolving power have been described. RESULTS: MLVA31 was applied to characterize the French National Reference Laboratory collection. The total collection of 130 strains is resolved in 35 genotypes. The 119 veterinary and environmental strains collection in France were resolved into 26 genotypes belonging to three canSNP lineages and four MLVA clonal complexes (CCs with particular geographical clustering. A subset of seven loci (MLVA7 is proposed to constitute a first line assay. The loci are compatible with moderate resolution equipment such as agarose gel electrophoresis and show a good congruence value with MLVA31. The associated MLVA and SNP data was imported together with published genotyping data by taking advantage of major enhancements to the MLVAbank software and web site. CONCLUSIONS: The present report provides a wide coverage of the genetic diversity of naturally occurring B. anthracis strains in France as can be revealed by MLVA. The data obtained suggests that once such coverage is achieved, it becomes possible to devise optimized first-line MLVA assays comprising a sufficiently low number of loci to be typed either in one multiplex PCR or on agarose gels. Such a selection of seven loci is proposed here, and future similar investigations in additional countries will indicate to which extend the same selection can be used worldwide as a common minimum set. It is hoped that this approach will contribute to an efficient and low-cost routine surveillance of important pathogens for biosecurity such as

  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.

  8. The poly-γ-d-glutamic acid capsule surrogate of the Bacillus anthracis capsule induces nitric oxide production via the platelet activating factor receptor signaling pathway.

    Science.gov (United States)

    Lee, Hae-Ri; Jeon, Jun Ho; Park, Ok-Kyu; Chun, Jeong-Hoon; Park, Jungchan; Rhie, Gi-Eun

    2015-12-01

    The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, confers protection of the bacillus from phagocytosis and allows its unimpeded growth in the host. PGA capsules released from B. anthracis are associated with lethal toxin in the blood of experimentally infected animals and enhance the cytotoxic effect of lethal toxin on macrophages. In addition, PGA capsule itself activates macrophages and dendritic cells to produce proinflammatory cytokine such as IL-1β, indicating multiple roles of PGA capsule in anthrax pathogenesis. Here we report that PGA capsule of Bacillus licheniformis, a surrogate of B. anthracis capsule, induces production of nitric oxide (NO) in RAW264.7 cells and bone marrow-derived macrophages. NO production was induced by PGA in a dose-dependent manner and was markedly reduced by inhibitors of inducible NO synthase (iNOS), suggesting iNOS-dependent production of NO. Induction of NO production by PGA was not observed in macrophages from TLR2-deficient mice and was also substantially inhibited in RAW264.7 cells by pretreatment of TLR2 blocking antibody. Subsequently, the downstream signaling events such as ERK, JNK and p38 of MAPK pathways as well as NF-κB activation were required for PGA-induced NO production. In addition, the induced NO production was significantly suppressed by treatment with antagonists of platelet activating factor receptor (PAFR) or PAFR siRNA, and mediated through PAFR/Jak2/STAT-1 signaling pathway. These findings suggest that PGA capsule induces NO production in macrophages by triggering both TLR2 and PAFR signaling pathways which lead to activation of NF-kB and STAT-1, respectively.

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

    Science.gov (United States)

    Ndumnego, Okechukwu C; Köhler, Susanne M; Crafford, Jannie; van Heerden, Henriette; Beyer, Wolfgang

    2016-10-01

    The Sterne 34F2 live spore vaccine (SLSV) developed in 1937 is the most widely used veterinary vaccine against anthrax. However, literature on the immunogenicity of this vaccine in a target ruminant host is scarce. In this study, we evaluated the humoral response to the Bacillus anthracis protective antigen (rPA), a recombinant bacillus collagen-like protein of anthracis (rBclA), formaldehyde inactivated spores (FIS) prepared from strain 34F2 and a vegetative antigen formulation prepared from a capsule and toxin deficient strain (CDC 1014) in Boer goats. The toxin neutralizing ability of induced antibodies was evaluated using an in vitro toxin neutralization assay. The protection afforded by the vaccine was also assessed in vaccinates. Anti-rPA, anti-FIS and lethal toxin neutralizing titres were superior after booster vaccinations, compared to single vaccinations. Qualitative analysis of humoral responses to rPA, rBclA and FIS antigens revealed a preponderance of anti-FIS IgG titres following either single or double vaccinations with the SLSV. Antibodies against FIS and rPA both increased by 350 and 300-fold following revaccinations respectively. There was no response to rBclA following vaccinations with the SLSV. Toxin neutralizing titres increased by 80-fold after single vaccination and 700-fold following a double vaccination. Lethal challenge studies in naïve goats indicated a minimum infective dose of 36 B. anthracis spores. Single and double vaccination with the SLSV protected 4/5 and 3/3 of goats challenged with>800 spores respectively. An early booster vaccination following the first immunization is suggested in order to achieve a robust immunity. Results from this study indicate that this crucial second vaccination can be administered as early as 3 months after the initial vaccination.

  10. Ecological recovery in ERA

    DEFF Research Database (Denmark)

    EFSA Scientific Committee (Scientific Committee); Topping, Christopher John

    2016-01-01

    EFSA performs environmental risk assessments (ERAs) for single potential stressors such as plant protection products, genetically modified organisms and feed additives and for invasive alien species that are harmful for plant health. In this risk assessment domain, the EFSA Scientific Committee...... recognises the importance of more integrated ERAs considering both the local and landscape scales, as well as the possible co-occurrence of multiple potential stressors that fall under the remit of EFSA, which are important when addressing ecological recovery. In this scientific opinion, the Scientific...... Committee gathered scientific knowledge on the potential for the recovery of non-target organisms for the further development of ERA. Current EFSA guidance documents and opinions were reviewed on how ecological recovery is addressed in ERA schemes. In addition, this scientific opinion is based on expert...

  11. Chasing Ecological Interactions

    Science.gov (United States)

    2016-01-01

    Basic research on biodiversity has concentrated on individual species—naming new species, studying distribution patterns, and analyzing their evolutionary relationships. Yet biodiversity is more than a collection of individual species; it is the combination of biological entities and processes that support life on Earth. To understand biodiversity we must catalog it, but we must also assess the ways species interact with other species to provide functional support for the Tree of Life. Ecological interactions may be lost well before the species involved in those interactions go extinct; their ecological functions disappear even though they remain. Here, I address the challenges in studying the functional aspects of species interactions and how basic research is helping us address the fast-paced extinction of species due to human activities. PMID:27631692

  12. Fundamental ecology is fundamental.

    Science.gov (United States)

    Courchamp, Franck; Dunne, Jennifer A; Le Maho, Yvon; May, Robert M; Thébaud, Christophe; Hochberg, Michael E

    2015-01-01

    The primary reasons for conducting fundamental research are satisfying curiosity, acquiring knowledge, and achieving understanding. Here we develop why we believe it is essential to promote basic ecological research, despite increased impetus for ecologists to conduct and present their research in the light of potential applications. This includes the understanding of our environment, for intellectual, economical, social, and political reasons, and as a major source of innovation. We contend that we should focus less on short-term, objective-driven research and more on creativity and exploratory analyses, quantitatively estimate the benefits of fundamental research for society, and better explain the nature and importance of fundamental ecology to students, politicians, decision makers, and the general public. Our perspective and underlying arguments should also apply to evolutionary biology and to many of the other biological and physical sciences.

  13. Biosemiotics and ecological monitoring

    DEFF Research Database (Denmark)

    Bruni, Luis Emilio

    2001-01-01

    Through recent decades a global cultural-institutional network has gradually grown up to project, implement and use the enormous technological web that is supposed to observe, monitor, communicate, inventory and asses our environment and its biodiversity in order to implement sustainable management...... models. The majority of “knowledge tools” that have been incorporated in the mainstream of this “techno-web” are amply based on a combination of mechanistic biology, genetic reductionism, economical determinism and neo-Darwinian cultural and biological perspectives. These approaches leave aside many...... of the qualitative and relational aspects that can only be grasped by considering the semiotic networks operative in complex ecological and cultural systems. In this paper, it is suggested that a biosemiotic approach to ecology may prove useful for the modelling process, which in turn will allow the construction...

  14. Editorial: Pedagogical Media Ecologies

    Directory of Open Access Journals (Sweden)

    Dorothee M. Meister

    2014-07-01

    Full Text Available From educational gaming through portable e-readers to cell phones, media are interpenetrating educational spaces and activities. Accordingly, understanding media in environmental or ecological terms has become increasingly important for education internationally. In North America, for example, the centenary of McLuhan’s birth has focused attention on approaches to media – whether oral, textual, electronic or digital– as a kind of environment in which education takes place. In parts of Europe, the so-called mediatic turn – following on the linguistic and iconic turns – has similarly emphasized the role of media as a condition for the possibility of educational activities and programs. With a few exceptions1 the papers in this special issue were first presented at the conference «Educational Media Ecologies: International Perspectives» which took place at the University of Paderborn, Germany, on March 27–28, 2012.2 The event was an interdisciplinary and transatlantic endeavor to bring together a wide range of perspectives on various issues relevant to educational media ecologies,3 and on related debates on mediation, medialization, mediatization, and mediality.4 The purpose of this volume, like the conference, is to foster and deepen international dialogue in the area of educational media. Areas of research and scholarship relevant to this dialogue include educational media, media literacy, educational philosophy, and media and cultural studies. The contributions, described below, put conceptual issues as well as social practices and applications at the center of the debate. Klaus Rummler opens the issue by clarifying the concept of ecology itself. Referencing a range of work over the past 50 years, Rummler describes how ecological models have been cast in sociological, semiotic, cultural, mediatic and other terms, and he explains the implications of these various perspectives for the study of educational contexts. Rummler also

  15. To the Ecological Aesthetics

    Institute of Scientific and Technical Information of China (English)

    王秀青

    2014-01-01

    Landscape design is one of the space form and space environment comprehensive planning and design, to im-prove the quality of urban environment, quality of life and urban landscape level play a very important role.It is expounded in this article based on the value of landscape ecological aesthetics under the background of productive landscape, discusses the"bigfoot aesthetics"innovative application in modern landscape design and new thinking.

  16. To the Ecological Aesthetics

    Institute of Scientific and Technical Information of China (English)

    王秀青

    2014-01-01

    Landscape design is one of the space form and space environment comprehensive planning and design, to im- prove the quality of urban environment, quality of life and urban landscape level play a very important role.It is expounded in this article based on the value of landscape ecological aesthetics under the background of productive landscape, discusses the "bigfoot aesthetics" innovative application in modern landscape design and new thinking.

  17. The Genome Sequence of Bacillus cereus ATCC 10987 Reveals Metabolic Adaptations and a Large Plasmid Related to Bacillus anthracis pXO1

    Science.gov (United States)

    2004-01-01

    R.L. and Waites,K.B. (2003) Bacillus cereus bacteremia in a preterm neonate. J. Clin. Microbiol., 41, 3441±3444. 9. Ginsburg,A.S., Salazar,L.G., True... bacteremia and pneumonia due to Bacillus cereus . J. Clin. Microbiol., 35, 504±507. 12. Okinaka,R., Cloud,K., Hampton,O., Hoffmaster,A., Hill,K., Keim,P...The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1 David A. Rasko

  18. Proteomics Reveals that Proteins Expressed During the Early Stage of Bacillus anthracis Infection Are Potential Targets for the Development of Vaccines and Drugs

    Institute of Scientific and Technical Information of China (English)

    Chun-Ming Huang; Craig A. Elmets; De-chu C. Tang; Fuming Li; Nabiha Yusuf

    2004-01-01

    In this review, we advance a new concept in developing vaccines and/or drugs to target specific proteins expressed during the early stage of Bacillus anthracis (an thrax) infection and address existing challenges to this concept. Three proteins (immune inhibitor A, GPR-like spore protease, and alanine racemase) initially identified by proteomics in our laboratory were found to have differential expres sions during anthrax spore germination and early outgrowth. Other studies of different bacillus strains indicate that these three proteins are involved in either germination or cytotoxicity of spores, suggesting that they may serve as potential targets for the design of anti-anthrax vaccines and drugs.

  19. Discrimination of bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray.

    Energy Technology Data Exchange (ETDEWEB)

    Bavykin, S. G.; Mikhailovich, V. M.; Zakharyev, V. M.; Lysov, Y. P.; Kelly, J. J.; Alferov, O. S.; Jackman, J.; Stahl, D. A.; Mirzabekov, A. D.; Gavin, I. M.; Kukhtin, A. V.; Chandler, D. (Biochip Technology Center); (Engelhardt Inst. of Molecular Biology); (Northwestern Univ.); (Georgetown Univ.)

    2008-01-30

    Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5 min. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect

  20. The Poly-γ-d-Glutamic Acid Capsule Surrogate of the Bacillus anthracis Capsule Is a Novel Toll-Like Receptor 2 Agonist.

    Science.gov (United States)

    Jeon, Jun Ho; Lee, Hae-Ri; Cho, Min-Hee; Park, Ok-Kyu; Park, Jungchan; Rhie, Gi-eun

    2015-10-01

    Bacillus anthracis is a pathogenic Gram-positive bacterium that causes a highly lethal infectious disease, anthrax. The poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors of B. anthracis, along with exotoxins. PGA enables B. anthracis to escape phagocytosis and immune surveillance. Our previous study showed that PGA activates the human macrophage cell line THP-1 and human dendritic cells, resulting in the production of the proinflammatory cytokine interleukin-1β (IL-1β) (M. H. Cho et al., Infect Immun 78:387-392, 2010, http://dx.doi.org/10.1128/IAI.00956-09). Here, we investigated PGA-induced cytokine responses and related signaling pathways in mouse bone marrow-derived macrophages (BMDMs) using Bacillus licheniformis PGA as a surrogate for B. anthracis PGA. Upon exposure to PGA, BMDMs produced proinflammatory mediators, including tumor necrosis factor alpha (TNF-α), IL-6, IL-12p40, and monocyte chemoattractant protein 1 (MCP-1), in a concentration-dependent manner. PGA stimulated Toll-like receptor 2 (TLR2) but not TLR4 in Chinese hamster ovary cells expressing either TLR2 or TLR4. The ability of PGA to induce TNF-α and IL-6 was retained in TLR4(-/-) but not TLR2(-/-) BMDMs. Blocking experiments with specific neutralizing antibodies for TLR1, TLR6, and CD14 showed that TLR6 and CD14 also were necessary for PGA-induced inflammatory responses. Furthermore, PGA enhanced activation of mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-κB), which are responsible for expression of proinflammatory cytokines. Additionally, PGA-induced TNF-α production was abrogated not only in MyD88(-/-) BMDMs but also in BMDMs pretreated with inhibitors of MAP kinases and NF-κB. These results suggest that immune responses induced by PGA occur via TLR2, TLR6, CD14, and MyD88 through activation of MAP kinase and NF-κB pathways.

  1. The Bacillus anthracis arylamine N-acetyltransferase ((BACAN)NAT1) that inactivates sulfamethoxazole, reveals unusual structural features compared with the other NAT isoenzymes

    DEFF Research Database (Denmark)

    Pluvinage, Benjamin; Li de la Sierra-Gallay, Inés; Kubiak, Xavier Jean Philippe

    2011-01-01

    Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that biotransform arylamine drugs. The Bacillus anthracis (BACAN)NAT1 enzyme affords increased resistance to the antibiotic sulfamethoxazole through its acetylation. We report the structure of (BACAN)NAT1. Unexpectedly...... marked differences in the mode of binding and location of coenzymeA when compared to the other NATs. This suggests that the mechanisms of cofactor recognition by NATs is more diverse than expected and supports the cofactor-binding site as being a unique subsite to target in drug design against bacterial...

  2. Resilience Through Ecological Network

    Directory of Open Access Journals (Sweden)

    Grazia Brunetta

    2014-05-01

    Full Text Available The paper explores the strategic role that urban biodiversity and ecosystem services management, natural infrastructure and adaptive governance approaches can play in making our economies and societies more resilient and in linking human societies and the natural environment. Resilience – a concept that entered the debate on urban governance – means the ability of urban systems, considered as linear-systems, to react to external disturbances by returning to some socio-ecological equilibrium steady-state by overcoming a crisis period (Gunderson & al. 2010, Newman & al. 2009. In this view, green infrastructures can assume a strategic role in restoring and enhancing the ecological and environmental livability in urban areas. Starting from the International and European context, the paper discusses innovative programs and interdisciplinary projects and practices (some cases in Turin Metropolitan Area to demonstrate how green infrastructures can increase the adaptive capacity of urban systems in term of resilience. They can contribute to increase the ability of European cities to adapt to climate change and to reduce their ecological footprints, to enhance security and life quality.

  3. Ecological planning: A review

    Science.gov (United States)

    Steiner, Frederick; Brooks, Kenneth

    1981-11-01

    Beginning with the passage of the National Environmental Policy Act in 1969, the federal government of the United States has enacted numerous pieces of legislation intended to protect or conserve the environment. Other national governments have also enacted environmental legislation during the past two decades. State and local governments have also adopted policies concerned with environmental planning and management. Multiple laws and overlapping governmental agency responsibilities have confused development and resource management efforts. A comprehensive methodology that integrates the legal mandates and the agency missions into a common and unified framework is needed. Ecological planning offers such a method. Application of the method allows planners and resource managers to better understand the nature and character of the land and/or resource and therefore make better decisions about its appropriate use or management. The steps taken in an ecological planning process—1) goal setting, 2) inventory and analysis of data, 3) suitability analysis, 4) developing alternatives, 5) implementation, 6) administration, and 7) evaluation—are outlined and explained. Hand-drawn overlays and computer programs as techniques for handling ecological planning information are compared. Observations and suggestions for further research are offered.

  4. Ecological Econophysics for Degrowth

    Directory of Open Access Journals (Sweden)

    Salvador Pueyo

    2014-05-01

    Full Text Available This paper outlines a synthesis of ecological economics with econophysics and other complexity approaches to economics. Arguably, the resulting “ecological econophysics” will be scientifically sounder than mainstream economics and much better suited to addressing a major challenge of our times: the development of democratically-based policies to reduce economic throughput to an environmentally sustainable level without triggering economic crises and without excluding part of the world’s population, i.e., to implement degrowth. Degrowth will need major structural changes, which leads us to question whether there are limits to the malleability of the economic system’s architecture. A fundamental limit will be encountered if, as suggested by the physics of complexity, long-lasting complex systems always occur midway between an ordered and a disordered state. There is much evidence that this hypothesis holds and that the current economic system satisfies this condition. However, this does not mean that the problems posed by this system should be unavoidable. Ecological econophysics gives clues to identifying alternative economic systems that would also function between order and chaos, but which would have radically different implications for environmental sustainability and social justice.

  5. Research on Ecological Civilization Evaluation Index System

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Ecological civilization, which refers to the harmonious development of man and the nature, has the connotation of the ecological material civilization, ecological consciousness civilization, ecological institutional civilization and ecological behavior civilization. The research on ecological civilization evaluation index system is important in that it can provide the guidance for the construction of ecological civilization, and besides it can improve public recognition of the importance of ecological civil...

  6. Statistical ecology comes of age.

    Science.gov (United States)

    Gimenez, Olivier; Buckland, Stephen T; Morgan, Byron J T; Bez, Nicolas; Bertrand, Sophie; Choquet, Rémi; Dray, Stéphane; Etienne, Marie-Pierre; Fewster, Rachel; Gosselin, Frédéric; Mérigot, Bastien; Monestiez, Pascal; Morales, Juan M; Mortier, Frédéric; Munoz, François; Ovaskainen, Otso; Pavoine, Sandrine; Pradel, Roger; Schurr, Frank M; Thomas, Len; Thuiller, Wilfried; Trenkel, Verena; de Valpine, Perry; Rexstad, Eric

    2014-12-01

    The desire to predict the consequences of global environmental change has been the driver towards more realistic models embracing the variability and uncertainties inherent in ecology. Statistical ecology has gelled over the past decade as a discipline that moves away from describing patterns towards modelling the ecological processes that generate these patterns. Following the fourth International Statistical Ecology Conference (1-4 July 2014) in Montpellier, France, we analyse current trends in statistical ecology. Important advances in the analysis of individual movement, and in the modelling of population dynamics and species distributions, are made possible by the increasing use of hierarchical and hidden process models. Exciting research perspectives include the development of methods to interpret citizen science data and of efficient, flexible computational algorithms for model fitting. Statistical ecology has come of age: it now provides a general and mathematically rigorous framework linking ecological theory and empirical data.

  7. A One Health, participatory epidemiology assessment of anthrax (Bacillus anthracis) management in Western Uganda.

    Science.gov (United States)

    Coffin, Jeanne L; Monje, Fred; Asiimwe-Karimu, Grace; Amuguni, Hellen Janetrix; Odoch, Terence

    2015-03-01

    Sporadic anthrax outbreaks have occurred in and around Uganda's Queen Elizabeth National Park (QENP) for years, affecting wildlife, domestic animals, and humans. Reported outbreaks (2004-2005 and 2010) in QENP collectively killed over 500 wild animals and over 400 domestic animals. A 2011 outbreak in Sheema district temporarily froze local markets while killing two humans and seven bovines. One Health is multidisciplinary at its core, yet studies sometimes focus on the effects of animals on human health to the detriment of investigating the surrounding ecological and cultural contexts. Participatory methods connect problems - such as disease - to their context. A multidisciplinary team used participatory epidemiology and conventional structured questionnaires to investigate the impacts of anthrax on human livelihoods and the related perceptions of conservation, public health, and veterinary health efforts in the QENP area. Proximities to previous anthrax outbreaks and to QENP were treated as risk factors in the collection and evaluation of data. Participants' feedback indicates that anthrax prevalence may be greater than officially reported. Community member perceptions about anthrax and other diseases appear to be more closely related to their proximity to QENP than their proximity to anthrax outbreaks. Neither risk factor had a strong effect on knowledge of disease, nor any effect on behaviors associated with disease response or control. Instead, participants reported that social pressures, the economics of poverty, and the lack of health and veterinary infrastructure highly influenced responses to disease. The complex connections between the social needs and the economic context of these communities seem to be undermining current anthrax control and education measures. This livelihood-based decision-making may be unlikely to respond to educational intervention alone. This study provides a strong base for further research and for improvements in effective disease

  8. Identification and Analysis of Novel Amino-Acid Sequence Repeats in Bacillus anthracis str. Ames Proteome Using Computational Tools

    Directory of Open Access Journals (Sweden)

    D. Satyanarayana Rao

    2007-02-01

    Full Text Available We have identified four repeats and ten domains that are novel in proteins encoded by the Bacillus anthracis str. Ames proteome using automated in silico methods. A “repeat” corresponds to a region comprising less than 55-amino-acid residues that occur more than once in the protein sequence and sometimes present in tandem. A “domain” corresponds to a conserved region with greater than 55-amino-acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1 57-amino-acid-residue PxV domain, (2 122-amino-acid-residue FxF domain, (3 111-amino-acid-residue YEFF domain, (4 109-amino-acid-residue IMxxH domain, (5 103-amino-acid-residue VxxT domain, (6 84-amino-acid-residue ExW domain, (7 104-amino-acid-residue NTGFIG domain, (8 36-amino-acid-residue NxGK repeat, (9 95-amino-acid-residue VYV domain, (10 75-amino-acid-residue KEWE domain, (11 59-amino-acid-residue AFL domain, (12 53-amino-acid-residue RIDVK repeat, (13 (a 41-amino-acid-residue AGQF repeat and (b 42-amino-acid-residue GSAL repeat. A repeat or domain type is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure.

  9. Rapid identification of genetic modifications in Bacillus anthracis using whole genome draft sequences generated by 454 pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Peter E Chen

    Full Text Available BACKGROUND: The anthrax letter attacks of 2001 highlighted the need for rapid identification of biothreat agents not only for epidemiological surveillance of the intentional outbreak but also for implementing appropriate countermeasures, such as antibiotic treatment, in a timely manner to prevent further casualties. It is clear from the 2001 cases that survival may be markedly improved by administration of antimicrobial therapy during the early symptomatic phase of the illness; i.e., within 3 days of appearance of symptoms. Microbiological detection methods are feasible only for organisms that can be cultured in vitro and cannot detect all genetic modifications with the exception of antibiotic resistance. Currently available immuno or nucleic acid-based rapid detection assays utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel natural variations or intentional genetic modifications that circumvent the targets of the detection assays or in the case of a biological attack using an antibiotic resistant or virulence enhanced Bacillus anthracis, to advise on therapeutic treatments. METHODOLOGY/PRINCIPAL FINDINGS: We show here that the Roche 454-based pyrosequencing can generate whole genome draft sequences of deep and broad enough coverage of a bacterial genome in less than 24 hours. Furthermore, using the unfinished draft sequences, we demonstrate that unbiased identification of known as well as heretofore-unreported genetic modifications that include indels and single nucleotide polymorphisms conferring antibiotic and phage resistances is feasible within the next 12 hours. CONCLUSIONS/SIGNIFICANCE: Second generation sequencing technologies have paved the way for sequence-based rapid identification of both known and previously undocumented genetic modifications in cultured, conventional and newly emerging biothreat agents. Our findings have significant implications in

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

    Energy Technology Data Exchange (ETDEWEB)

    Grecz, N.; Kang, T.

    1973-01-01

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

  11. Ecological and general systems an introduction to systems ecology

    CERN Document Server

    Odum, Howard T.

    1994-01-01

    Using an energy systems language that combines energetics, kinetics, information, cybernetics, and simulation, Ecological and General Systems compares models of many fields of science, helping to derive general systems principles. First published as Systems Ecology in 1983, Ecological and General Systems proposes principles of self-organization and the designs that prevail by maximizing power and efficiency. Comparisons to fifty other systems languages are provided. Innovative presentations are given on earth homeostasis (Gaia); the inadequacy of presenting equations without network relationships and energy constraints; the alternative interpretation of high entropy complexity as adaptive structure; basic equations of ecological economics; and the energy basis of scientific hierarchy.

  12. Ecological model of extinctions

    CERN Document Server

    Abramson, G

    1997-01-01

    We present numerical results based on a simplified ecological system in evolution, showing features of extinction similar to that claimed for the biosystem on Earth. In the model each species consists of a population in interaction with the others, that reproduces and evolves in time. Each species is simultaneously a predator and a prey in a food chain. Mutations that change the interactions are supposed to occur randomly at a low rate. Extinctions of populations result naturally from the predator-prey dynamics. The model is not pinned in a fitness variable, and natural selection arises from the dynamics.

  13. Ecology Beyond Building

    DEFF Research Database (Denmark)

    Peters, Terri

    2011-01-01

    As the designers of the WWf building in Zeist, The Netherslands a CO2-neutral, self-sufficient office complex, RAU has set the bar for sustainable research and design. Guesteditor Terri Peters visited the firm's studio in Amsterdam to talk to principal Thomas Rau. As Peters relates, Rau prefers t...... to put on the dwindling supply of raw materials rather than the immidiate problems of energy consumption for which there are solutions within reach. With the emphasis on a more far-reaching approach, he places buildings in a wider context of ecological thinking and systems....

  14. Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

    Directory of Open Access Journals (Sweden)

    Matthew D Reed

    Full Text Available Protective antigen (PA, one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax. Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel, elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg.

  15. Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

    Science.gov (United States)

    Reed, Matthew D; Wilder, Julie A; Mega, William M; Hutt, Julie A; Kuehl, Philip J; Valderas, Michelle W; Chew, Lawrence L; Liang, Bertrand C; Squires, Charles H

    2015-01-01

    Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg.

  16. Recombinant expression of Bacillus anthracis lethal toxin components of Indian isolate in Escherichia coli and determination of its acute toxicity level in mouse model.

    Science.gov (United States)

    Nagendra, Suryanarayana; Vanlalhmuaka; Verma, Sarika; Tuteja, Urmil; Thavachelvam, Kulanthaivel

    2015-12-15

    Bacillus anthracis lethal toxin (LeTx) is the principle factor responsible for toxaemia and anthrax related death. Lethal toxin consist of two proteins viz protective antigen (PA) and lethal factor which combines in a typical fashion similar to other toxins belonging to A-B toxin super family. The amount of LeTx required to kill a particular organism generally differs among strains owing to their geographical distributions and genetic variation. In the present study, we have cloned PA and LF genes from B. anthracis clinical isolate of Indian origin and expressed them in soluble form employing Escherichia coli expression system. Both the proteins were purified to near homogeneity level using Immobilized metal ion affinity chromatography (IMAC). Further we have used equal ratio of both the proteins to form LeTx and determined its acute toxicity level in Balb/c mice by graphical method of Miller and Tainter. The LD50 value of LeTx by intravenous (i.v) route was found to be 0.97 ± 0.634 mg kg(-1) Balb/c mice. This study highlights the expression of recombinant LeTx from E. coli and assessing its acute toxicity level in experimental mouse model.

  17. Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

    Science.gov (United States)

    Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

    2014-10-01

    Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages.

  18. Structure of the Bacillus anthracis Sortase A Enzyme Bound to Its Sorting Signal: A FLEXIBLE AMINO-TERMINAL APPENDAGE MODULATES SUBSTRATE ACCESS.

    Science.gov (United States)

    Chan, Albert H; Yi, Sung Wook; Terwilliger, Austen L; Maresso, Anthony W; Jung, Michael E; Clubb, Robert T

    2015-10-16

    The endospore forming bacterium Bacillus anthracis causes lethal anthrax disease in humans and animals. The ability of this pathogen to replicate within macrophages is dependent upon the display of bacterial surface proteins attached to the cell wall by the B. anthracis Sortase A ((Ba)SrtA) enzyme. Previously, we discovered that the class A (Ba)SrtA sortase contains a unique N-terminal appendage that wraps around the body of the protein to contact the active site of the enzyme. To gain insight into its function, we determined the NMR structure of (Ba)SrtA bound to a LPXTG sorting signal analog. The structure, combined with dynamics, kinetics, and whole cell protein display data suggest that the N terminus modulates substrate access to the enzyme. We propose that it may increase the efficiency of protein display by reducing the unproductive hydrolytic cleavage of enzyme-protein covalent intermediates that form during the cell wall anchoring reaction. Notably, a key active site loop (β7/β8 loop) undergoes a disordered to ordered transition upon binding the sorting signal, potentially facilitating recognition of lipid II.

  19. Sensory Ecology, Evolution, and Behavior

    Institute of Scientific and Technical Information of China (English)

    Martin STEVENS; Guest Editor

    2010-01-01

    @@ 1 Introduction Sensory ecology deals with how animals capture in formation from their environment, and the sensory sys tems involved in doing so (Hailman, 1977; Lythgoe, 1979; Dusenbery, 1992; Mappes and Stevens 2010). Although the term sensory ecology itself is compara tively recent, its basis has a long history, in part due to numerous links with subjects such as neurobiology, physiology, ethology, and evolutionary behavioral ecology.

  20. ECOLOGY AFFECTED IN OIL EXPLOITATION

    Institute of Scientific and Technical Information of China (English)

    Wang Mingren; Zeng Dezhang; Zhang Shiquan; Shi Lifeng

    1997-01-01

    @@ Introduction During the Eighth Five-Year Plan period(1991-1995), a study about crude impacts on ecology in oil exploitation was conducted in seven representative onshore oil fields of China. The study discusses crude pollutant's impacts on ecology in terms of its production,movement, transformation and concentration in the ecological system,as well as its toxicity and damage degree on living things , by means of investigation on the spot, test analysis and analogue test.

  1. [Landscape and ecological genomics].

    Science.gov (United States)

    Tetushkin, E Ia

    2013-10-01

    Landscape genomics is the modern version of landscape genetics, a discipline that arose approximately 10 years ago as a combination of population genetics, landscape ecology, and spatial statistics. It studies the effects of environmental variables on gene flow and other microevolutionary processes that determine genetic connectivity and variations in populations. In contrast to population genetics, it operates at the level of individual specimens rather than at the level of population samples. Another important difference between landscape genetics and genomics and population genetics is that, in the former, the analysis of gene flow and local adaptations takes quantitative account of landforms and features of the matrix, i.e., hostile spaces that separate species habitats. Landscape genomics is a part of population ecogenomics, which, along with community genomics, is a major part of ecological genomics. One of the principal purposes of landscape genomics is the identification and differentiation of various genome-wide and locus-specific effects. The approaches and computation tools developed for combined analysis of genomic and landscape variables make it possible to detect adaptation-related genome fragments, which facilitates the planning of conservation efforts and the prediction of species' fate in response to expected changes in the environment.

  2. ECOLOGICAL GROWTH BOUNDARIES

    Directory of Open Access Journals (Sweden)

    Anna BLUSZCZ

    2017-01-01

    Full Text Available The trends of the society for the continuous growth, combined with the demographic changes, today have led to the important ecological problems on a global scale, which include, among others: the increased use of non-renewable natu-ral resources, an increase of the greenhouse gas emissions, contamination of soil, water, air and the progressive degra-dation of ecosystems. In the face of such serious threats the global initiatives of all countries are important to limit the results of the excessive consumption. The aim of the article is to present the methods of measurement of the consump-tion level of natural resources by the societies and the examination of relationships between the level of development of the societies and the use of resources. The popular measure – the ecological footprint – was used as a measurement method for the consumption of the today’s generations in relation to the regenerative possibilities of the natural envi-ronment. On the other hand, as the assessment method for the level of development of societies – the Human Develop-ment Index (HDI, including three basic areas: the life expectancy, GDP level per capita and education was used. The results of the research indicate that the current trend of the unlimited consumption of the highly developed countries takes place at the expense of the future generations.

  3. Ecological Growth Boundaries

    Science.gov (United States)

    Bluszcz, Anna

    2017-03-01

    The trends of the society for the continuous growth, combined with the demographic changes, today have led to the important ecological problems on a global scale, which include, among others: the increased use of non-renewable natural resources, an increase of the greenhouse gas emissions, contamination of soil, water, air and the progressive degradation of ecosystems. In the face of such serious threats the global initiatives of all countries are important to limit the results of the excessive consumption. The aim of the article is to present the methods of measurement of the consumption level of natural resources by the societies and the examination of relationships between the level of development of the societies and the use of resources. The popular measure - the ecological footprint - was used as a measurement method for the consumption of the today's generations in relation to the regenerative possibilities of the natural environment. On the other hand, as the assessment method for the level of development of societies - the Human Development Index (HDI), including three basic areas: the life expectancy, GDP level per capita and education was used. The results of the research indicate that the current trend of the unlimited consumption of the highly developed countries takes place at the expense of the future generations.

  4. ECOLOGICAL BUILDING DESIGN DETERMINANTS

    Directory of Open Access Journals (Sweden)

    Ali Vakili-Ardebili

    2010-11-01

    Full Text Available The sustainable building design process is driven on the basis of a range of design eco-indicators. Consideration of a multitude of eco-determinants, such as environment, economy, resources, energy consumption and society values in addition to design characteristics and contexts, makes the process of ecological design even more complex. A large number of eco-drivers are extracted from the literature and current design practices. To gain a better insight on eco-design determinants, a survey focusing on the use of eco-design drivers has been conducted with various architects in the UK. The factor analysis method was used to remove redundant data from the survey. Through the factor analysis approach, 115 eco-determinants are grouped into six main clusters. This article presents the process, analysis and findings of this work. The extracted eco-indicators and their associated clusters can be used to improve the process of ecological building design. DOI: 10.3763/aedm.2008.0096 Source: Architectural Engineering and Design Management, Volume 6, Number 2, 2010 , pp. 111-131(21

  5. Graduate Education in Ecological Economics

    OpenAIRE

    John M. Polimeni

    2004-01-01

    Interest in graduate education in ecological economics is increasing. However, no formal plan of study for a Ph.D. in ecological economics has been disseminated. The lack of a formal plan is problematic as the field of ecological economics matures, interest grows, and new programs are being developed. This paper attempts to fill a void by creating a program of study addressing the proficiencies a graduate student in ecological economics should have upon completion of his/her Ph.D. based on th...

  6. Ecological implications of behavioural syndromes.

    Science.gov (United States)

    Sih, Andrew; Cote, Julien; Evans, Mara; Fogarty, Sean; Pruitt, Jonathan

    2012-03-01

    Interspecific trait variation has long served as a conceptual foundation for our understanding of ecological patterns and dynamics. In particular, ecologists recognise the important role that animal behaviour plays in shaping ecological processes. An emerging area of interest in animal behaviour, the study of behavioural syndromes (animal personalities) considers how limited behavioural plasticity, as well as behavioural correlations affects an individual's fitness in diverse ecological contexts. In this article we explore how insights from the concept and study of behavioural syndromes provide fresh understanding of major issues in population ecology. We identify several general mechanisms for how population ecology phenomena can be influenced by a species or population's average behavioural type, by within-species variation in behavioural type, or by behavioural correlations across time or across ecological contexts. We note, in particular, the importance of behavioural type-dependent dispersal in spatial ecology. We then review recent literature and provide new syntheses for how these general mechanisms produce novel insights on five major issues in population ecology: (1) limits to species' distribution and abundance; (2) species interactions; (3) population dynamics; (4) relative responses to human-induced rapid environmental change; and (5) ecological invasions.

  7. Ecological Effects of Allelopathic Plants

    DEFF Research Database (Denmark)

    Kruse, M.; Strandberg, M.; Strandberg, B.

    with the environment through spread of GM-plants or transgenes outside agricultural areas. The last chapter discuss GM-allelopathic plants in relation to the ecological risk assessment. Preface: This report is based on a literature review on allelopathy from an ecological impact point of view carried out in 1999...... on allelopathy in these crops. It discusses the ecological effects of allelopathic plants in natural ecosystems and factors of importance for the effects of these plants are pointed out. Finally the report presents suggestions for an ecological risk assessment of crops with an enhanced release of allelochemicals...

  8. Reinventing the Wheel: Teaching Restoration Ecology without the Ecology

    Science.gov (United States)

    Speldewinde, Peter

    2010-01-01

    Restoration ecology is "the process of assisting the recovery of an ecosystem that has been degraded, damaged or destroyed." Restoration can range from returning the system to its "natural" state through to restoring some ecological functionality to a system. The University of Western Australia offers an undergraduate degree in Restoration…

  9. Civic Ecology: Linking Social and Ecological Approaches in Extension

    Science.gov (United States)

    Krasny, Marianne E.; Tidball, Keith G.

    2010-01-01

    Civic ecology refers to the philosophy and science of community forestry, community gardening, watershed enhancement, and other volunteer-driven restoration practices in cities and elsewhere. Such practices, although often viewed as initiatives to improve a degraded environment, also foster social attributes of resilient social-ecological systems,…

  10. Reinventing the Wheel: Teaching Restoration Ecology without the Ecology

    Science.gov (United States)

    Speldewinde, Peter

    2010-01-01

    Restoration ecology is "the process of assisting the recovery of an ecosystem that has been degraded, damaged or destroyed." Restoration can range from returning the system to its "natural" state through to restoring some ecological functionality to a system. The University of Western Australia offers an undergraduate degree in…

  11. Ecological Research Division Theoretical Ecology Program. [Contains abstracts

    Energy Technology Data Exchange (ETDEWEB)

    1990-10-01

    This report presents the goals of the Theoretical Ecology Program and abstracts of research in progress. Abstracts cover both theoretical research that began as part of the terrestrial ecology core program and new projects funded by the theoretical program begun in 1988. Projects have been clustered into four major categories: Ecosystem dynamics; landscape/scaling dynamics; population dynamics; and experiment/sample design.

  12. Statistical physics and ecology

    Science.gov (United States)

    Volkov, Igor

    This work addresses the applications of the methods of statistical physics to problems in population ecology. A theoretical framework based on stochastic Markov processes for the unified neutral theory of biodiversity is presented and an analytical solution for the distribution of the relative species abundance distribution both in the large meta-community and in the small local community is obtained. It is shown that the framework of the current neutral theory in ecology can be easily generalized to incorporate symmetric density dependence. An analytically tractable model is studied that provides an accurate description of beta-diversity and exhibits novel scaling behavior that leads to links between ecological measures such as relative species abundance and the species area relationship. We develop a simple framework that incorporates the Janzen-Connell, dispersal and immigration effects and leads to a description of the distribution of relative species abundance, the equilibrium species richness, beta-diversity and the species area relationship, in good accord with data. Also it is shown that an ecosystem can be mapped into an unconventional statistical ensemble and is quite generally tuned in the vicinity of a phase transition where bio-diversity and the use of resources are optimized. We also perform a detailed study of the unconventional statistical ensemble, in which, unlike in physics, the total number of particles and the energy are not fixed but bounded. We show that the temperature and the chemical potential play a dual role: they determine the average energy and the population of the levels in the system and at the same time they act as an imbalance between the energy and population ceilings and the corresponding average values. Different types of statistics (Boltzmann, Bose-Einstein, Fermi-Dirac and one corresponding to the description of a simple ecosystem) are considered. In all cases, we show that the systems may undergo a first or a second order

  13. An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis.

    Science.gov (United States)

    Drum, C L; Yan, S Z; Sarac, R; Mabuchi, Y; Beckingham, K; Bohm, A; Grabarek, Z; Tang, W J

    2000-11-17

    The edema factor exotoxin produced by Bacillus anthracis is an adenylyl cyclase that is activated by calmodulin (CaM) at resting state calcium concentrations in infected cells. A C-terminal 60-kDa fragment corresponding to the catalytic domain of edema factor (EF3) was cloned, overexpressed in Escherichia coli, and purified. The N-terminal 43-kDa domain (EF3-N) of EF3, the sole domain of edema factor homologous to adenylyl cyclases from Bordetella pertussis and Pseudomonas aeruginosa, is highly resistant to protease digestion. The C-terminal 160-amino acid domain (EF3-C) of EF3 is sensitive to proteolysis in the absence of CaM. The addition of CaM protects EF3-C from being digested by proteases. EF3-N and EF3-C were expressed separately, and both fragments were required to reconstitute full CaM-sensitive enzyme activity. Fluorescence resonance energy transfer experiments using a double-labeled CaM molecule were performed and indicated that CaM adopts an extended conformation upon binding to EF3. This contrasts sharply with the compact conformation adopted by CaM upon binding myosin light chain kinase and CaM-dependent protein kinase type II. Mutations in each of the four calcium binding sites of CaM were examined for their effect on EF3 activation. Sites 3 and 4 were found critical for the activation, and neither the N- nor the C-terminal domain of CaM alone was capable of activating EF3. A genetic screen probing loss-of-function mutations of EF3 and site-directed mutations based on the homology of the edema factor family revealed a conserved pair of aspartate residues and an arginine that are important for catalysis. Similar residues are essential for di-metal-mediated catalysis in mammalian adenylyl cyclases and a family of DNA polymerases and nucleotidyltransferases. This suggests that edema factor may utilize a similar catalytic mechanism.

  14. Socio-Ecological Innovation

    DEFF Research Database (Denmark)

    Edgeman, Rick; Eskildsen, Jacob Kjær

    for Sustainability is (generally) a subset of the enterprise innovation portfolio. Effective and efficient integration of Innovation for Sustainability and Sustainable Innovation aids translation of 3E (equity, ecology, economy) enterprise strategyof the Triple Top Line into 3P (people, planet, profit) Triple Bottom...... Line enterprise results in operationalization of the cradle‐to‐cradle product and service design, delivery, and lifecycle philosophy. While SEI is defined, it must also be assessed. Any serious assessment of SEI requires not only understanding of what it is, but also how it manifests and in what forms......, how developed or mature it is, and how future SEI strategy and results can be improved. As an aid to these efforts, non‐prescriptive approaches to SEI are discussed, maturity scale assessment for SEI is developed and discussed, and a simple assessment report that combines graphic and narrative...

  15. Information Retrieval for Ecological Syntheses

    Science.gov (United States)

    Bayliss, Helen R.; Beyer, Fiona R.

    2015-01-01

    Research syntheses are increasingly being conducted within the fields of ecology and environmental management. Information retrieval is crucial in any synthesis in identifying data for inclusion whilst potentially reducing biases in the dataset gathered, yet the nature of ecological information provides several challenges when compared with…

  16. Beijing Star Lake Ecology Park

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ Beijing Star Lake Ecology Park is a Five-star hotel which has developed multi-functions of restaurant, lodge, bath, landscape seeing, leisure,body exercise, recreation, Ecology agriculture,etc. Occupying an area of 500 mu, the park is an environmental friendly five-star hotel.

  17. Coastal Adaptation and Ecological Engineering

    Science.gov (United States)

    Cheong, S. M.

    2014-12-01

    Ecological engineering combines ecology and engineering to sustain coastal environment and facilitate adaptation to climate change. This paper discusses how the cases of mangroves, oyster reefs, and marshes help mainstream climate change with ecosystem conservation. It demonstrates the benefits of combining strategies to combat changing climate given the financial and political constraints.

  18. SRS ecology: Environmental information document

    Energy Technology Data Exchange (ETDEWEB)

    Wike, L.D.; Shipley, R.W.; Bowers, J.A. [and others

    1993-09-01

    The purpose of this Document is to provide a source of ecological information based on the exiting knowledge gained from research conducted at the Savannah River Site. This document provides a summary and synthesis of ecological research in the three main ecosystem types found at SRS and information on the threatened and endangered species residing there.

  19. Ecological Networks in Urban Landscapes

    NARCIS (Netherlands)

    Cook, E.A.

    2000-01-01

    This research focuses on the topic of ecological networks in urban landscapes. Analysis and planning of ecological networks is a relatively new phenomenon and is a response to fragmentation and deterioration of quality of natural systems. In agricultural areas and with existing nature preserves this

  20. Molecular ecology of aquatic microbes

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1994-12-31

    Abstracts of reports are presented from a meeting on Molecular Ecology of Aquatic Microbes. Topics included: opportunities offered to aquatic ecology by molecular biology; the role of aquatic microbes in biogeochemical cycles; characterization of the microbial community; the effect of the environment on aquatic microbes; and the targeting of specific biological processes.

  1. The dimensionality of ecological networks

    DEFF Research Database (Denmark)

    Eklöf, Anna; Jacob, Ute; Kopp, Jason;

    2013-01-01

    How many dimensions (trait-axes) are required to predict whether two species interact? This unanswered question originated with the idea of ecological niches, and yet bears relevance today for understanding what determines network structure. Here, we analyse a set of 200 ecological networks, incl...

  2. The Future of Urban Ecology

    DEFF Research Database (Denmark)

    Elle, Morten

    1998-01-01

    This article is discusing the basic conditions for a positive development of urban ecology in Denmark. A number of battles has to be won.......This article is discusing the basic conditions for a positive development of urban ecology in Denmark. A number of battles has to be won....

  3. Ecological Monitoring Information System (EMIS).

    Science.gov (United States)

    Fiene, Richard John; And Others

    A system for evaluating and monitoring child development projects, with possible computerization capabilities, was developed for the State of Pennsylvania in connection with 26 child development projects funded by the Appalachian Regional Commission. The Ecological Monitoring Information System (EMIS), provides a series of ecological measurement…

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

    Science.gov (United States)

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

    2008-06-01

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

  5. Multivalent Chromosomal Expression of the Clostridium botulinum Serotype A Neurotoxin Heavy-Chain Antigen and the Bacillus anthracis Protective Antigen in Lactobacillus acidophilus

    Science.gov (United States)

    Klaenhammer, Todd R.

    2016-01-01

    ABSTRACT Clostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of Lactobacillus acidophilus NCFM expressing from the chromosome (i) the nontoxic host receptor-binding domain of the heavy chain of Clostridium botulinum serotype A neurotoxin (BoNT/A-Hc), (ii) the anthrax protective antigen (PA), and (iii) both the BoNT/A-Hc and the PA. The BoNT/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from L. acidophilus and a dendritic-cell-targeting peptide. A chromosomal region downstream of lba0889 carrying a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed the heterologous expression of the two antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on the transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high-expression chromosomal regions. Levels of expression match high-copy-number plasmids and eliminate the requirement for antibiotic selective maintenance of recombinant plasmids. IMPORTANCE Clostridium botulinum and Bacillus anthracis produce potent neurotoxins that pose a biochemical warfare concern; therefore, effective vaccines against these bacteria are required. Chromosomal expression of antigens is

  6. 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.

  7. Social-Ecological Guilds: Putting People into Marine Historical Ecology

    Directory of Open Access Journals (Sweden)

    Janna M. Shackeroff

    2011-03-01

    Full Text Available Marine historical ecology provides historic insights into past ocean ecosystems that are crucial to effectively confronting the declining health and resilience in marine ecosystems. A more 'peopled' approach to marine historical ecology is necessary, given the heightened emphasis on human dimensions in marine management. This study examined the historical ecology of Hawaiian coral reef ecosystems through oral histories of diverse ocean experts, representing six traditional, local, and scientific knowledge systems. Based on 61 in-depth interviews with these ocean experts, historical trends, abundance, and distribution over 80 years and a 50-mile region for 271 species emerged. Analyzing trends by ecological guild, e.g., herbivores, proved inappropriate to these data; rather, based on qualitative analyses, five distinct trends encompassing nearly all species emerged in what we term "social-ecological guilds." Ocean expert's observations of change were surprisingly consistent, regardless of their knowledge system, whereas perceptions of change varied widely. The historical picture was far broader and richer when the contributions of six knowledge systems were incorporated, compared to that of any one alone. Social-ecological guilds also matter critically from a management perspective, because understanding how experts from a multiplicity of perspectives observe, interpret, and respond to ecological change can help managers anticipate responses to management activities and perhaps to design better management strategies.

  8. Ecology-driven stereotypes override race stereotypes.

    Science.gov (United States)

    Williams, Keelah E G; Sng, Oliver; Neuberg, Steven L

    2016-01-12

    Why do race stereotypes take the forms they do? Life history theory posits that features of the ecology shape individuals' behavior. Harsh and unpredictable ("desperate") ecologies induce fast strategy behaviors such as impulsivity, whereas resource-sufficient and predictable ("hopeful") ecologies induce slow strategy behaviors such as future focus. We suggest that individuals possess a lay understanding of ecology's influence on behavior, resulting in ecology-driven stereotypes. Importantly, because race is confounded with ecology in the United States, we propose that Americans' stereotypes about racial groups actually reflect stereotypes about these groups' presumed home ecologies. Study 1 demonstrates that individuals hold ecology stereotypes, stereotyping people from desperate ecologies as possessing faster life history strategies than people from hopeful ecologies. Studies 2-4 rule out alternative explanations for those findings. Study 5, which independently manipulates race and ecology information, demonstrates that when provided with information about a person's race (but not ecology), individuals' inferences about blacks track stereotypes of people from desperate ecologies, and individuals' inferences about whites track stereotypes of people from hopeful ecologies. However, when provided with information about both the race and ecology of others, individuals' inferences reflect the targets' ecology rather than their race: black and white targets from desperate ecologies are stereotyped as equally fast life history strategists, whereas black and white targets from hopeful ecologies are stereotyped as equally slow life history strategists. These findings suggest that the content of several predominant race stereotypes may not reflect race, per se, but rather inferences about how one's ecology influences behavior.

  9. Antibacterial activity of propolis against Bacillus anthracis%蜂胶对炭疽杆菌的抑菌活性

    Institute of Scientific and Technical Information of China (English)

    许兵红; 曾莉萍

    2011-01-01

    Objective To determine the antibacterial activity of propolis from Henan province against Bacillus anthracis.Methods The agar diffusion method was used to test for antibacterial activity. Antibacterial effects were measured after exposure to seven formulations of 31% propolis at pH ranging from 5.5-8.5 (5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5). Additionally, ten dilutions of propolis from 31% to 0.06% ( 1:512), including a 0 control, at pH 5.5 and a dose of 7 μl were examined in triplicate.Results The diameters (mm) of the zones of inhibition in each pH group, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5, were as follows:19.16 ± 0.29, 17.67 ± 0.29, 14.67 ± 0.58, 13.67 ± 0.29, 14.00 ± 0.00, 13.33 ± 1.15 and 12.00 ± 0.00 mm, respectively. The diameters in each concentration group, 15.50%, 7.75%, 3.88%, 1.94%, 0.97%, 0.48%, 0.24%, 0.12% and 0.06%, and the control group were: 19.50 ± 1.80, 19.67 ± 1.04, 17.75 ± 0.35, 15.67 ± 0.58, 14.00 ± 2.29, 13.17 ± 1.04, 11.83 ± 1.53, 10.83 ± 1.26,9.00±0.00 and 0.00±0.00 mm, respectively. Conclusion Propolis from Henan province had antibacterial activity against B. anthracis, which declined with increasing pH or decreasing concentration.%目的 了解产自河南省的蜂胶对炭疽杆菌的抑菌作用.方法 采用琼脂平板扩散法,将实验分为pH 5.5~8.5不同组(蜂胶浓度31%),以及在pH 5.5下设置10个不同浓度组,即将31%蜂胶溶液倍比稀释至0.06%(1∶512)及空白组(0),每组3次重复,每张滤纸片(φ6 mm)滴加蜂胶溶液7μl,记录蜂胶的抑菌效果.结果 河南省蜂胶在pH 5.5、6.0、6.5、7.0、7.5、8.0、8.5平板上的抑菌环直径依次为(19.16±0.29)、(17.67±0.29)、(14.67±0.58)、(13.67±0.29)、(14.00±0.00)、(13.33±1.15)、(12.00±0.00)mm,蜂胶浓度分别为15.50%、7.75%、3.88%、1.94%、0.97%、0.48%、0.24%、0.12%、0.06%和0(空白组)时,抑菌环直径依次为(19.50±1.80)、(19.67±1.04)、(17.75±0.35)、(15.67±0.58)、(14.00±2

  10. Missing ecology: integrating ecological perspectives with the social-ecological system framework

    Directory of Open Access Journals (Sweden)

    Graham Epstein

    2013-08-01

    Full Text Available The social-ecological systems framework was designed to provide a common research tool for interdisciplinary investigations of social-ecological systems. However, its origin in institutional studies of the commons belies its interdisciplinary ambitions and highlights its relatively limited attention to ecology and natural scientific knowledge. This paper considers the biophysical components of the framework and its epistemological foundations as it relates to the incorporation of knowledge from the natural sciences. It finds that the mixture of inductive and deductive reasoning associated with socially-oriented investigations of these systems is lacking on the ecological side, which relies upon induction alone. As a result the paper proposes the addition of a seventh core sub-system to the social-ecological systems framework, ecological rules, which would allow scholars to explicitly incorporate knowledge from the natural sciences for deductive reasoning. The paper shows, through an instructive case study, how the addition of ecological rules can provide a more nuanced description of the factors that contribute to outcomes in social-ecological systems.

  11. Emergence Unites Ecology and Society

    Directory of Open Access Journals (Sweden)

    Ronald L. Trosper

    2005-06-01

    Full Text Available The effort to combine analysis of ecosystems and social systems requires a firm theoretical basis. When humans are present in an ecosystem, their actions affect emergent structures; this paper examines forms of emergence that account for the presence of humans. Humans monitor and regulate ecosystems based on their cultural systems. Cultural systems consist of concepts linked in complicated ways that can form consistent world views, can contain inconsistencies, and may or may not accurately model the properties of a social–ecological system. Consequently, human monitoring and regulating processes will differ, depending on cultural systems. Humans, as agents, change or maintain pre-existing material and cultural emergent structures. The presentation is illustrated with a case study of fire-prone forests. The paper shows that explicit attention to emergence serves very well in unifying the following requirements for social–ecological analysis: coherent and observable definitions of sustainability; ways to link ecological and social phenomena; ways to understand cultural reasons for stability and instability in dynamic social–ecological systems; and ways to include human self-evaluation and culture within dynamic models of social–ecological systems. Analysis of cultural emergent structures clarifies many differences in assumptions among the fields of economics, sociology, political science, ecology, and ecological economics. Because it can be readily applied to empirical questions, the framework provides a good way to organize policy analysis that is not dominated by one or another discipline.

  12. Toward ecologically scaled landscape indices.

    Science.gov (United States)

    Vos, C C; Verboom, J; Opdam, P F; Ter Braak, C J

    2001-01-01

    Nature conservation is increasingly based on a landscape approach rather than a species approach. Landscape planning that includes nature conservation goals requires integrated ecological tools. However, species differ widely in their response to landscape change. We propose a framework of ecologically scaled landscape indices that takes into account this variation. Our approach is based on a combination of field studies of spatially structured populations (metapopulations) and model simulations in artificial landscapes. From these, we seek generalities in the relationship among species features, landscape indices, and metapopulation viability. The concept of ecological species profiles is used to group species according to characteristics that are important in metapopulations' response to landscape change: individual area requirements as the dominant characteristic of extinction risk in landscape patches and dispersal distance as the main determinant of the ability to colonize patches. The ecological profiles and landscape indices are then integrated into two ecologically scaled landscape indices (ESLI): average patch carrying capacity and average patch connectivity. The field data show that the fraction of occupied habitat patches is correlated with the two ESLI. To put the ESLI into a perspective of metapopulation persistence, we determine the viability for six ecological profiles at different degrees of habitat fragmentation using a metapopulation model and computer-generated landscapes. The model results show that the fraction of occupied patches is a good indicator for metapopulation viability. We discuss how ecological profiles, ESLI, and the viability threshold can be applied for landscape planning and design in nature conservation.

  13. Temporal ecology in the Anthropocene.

    Science.gov (United States)

    Wolkovich, E M; Cook, B I; McLauchlan, K K; Davies, T J

    2014-11-01

    Two fundamental axes - space and time - shape ecological systems. Over the last 30 years spatial ecology has developed as an integrative, multidisciplinary science that has improved our understanding of the ecological consequences of habitat fragmentation and loss. We argue that accelerating climate change - the effective manipulation of time by humans - has generated a current need to build an equivalent framework for temporal ecology. Climate change has at once pressed ecologists to understand and predict ecological dynamics in non-stationary environments, while also challenged fundamental assumptions of many concepts, models and approaches. However, similarities between space and time, especially related issues of scaling, provide an outline for improving ecological models and forecasting of temporal dynamics, while the unique attributes of time, particularly its emphasis on events and its singular direction, highlight where new approaches are needed. We emphasise how a renewed, interdisciplinary focus on time would coalesce related concepts, help develop new theories and methods and guide further data collection. The next challenge will be to unite predictive frameworks from spatial and temporal ecology to build robust forecasts of when and where environmental change will pose the largest threats to species and ecosystems, as well as identifying the best opportunities for conservation.

  14. PlcR在炭疽芽胞杆菌A16R中的功能研究%The function of PlcR inBacillus anthracis vaccine strain A16R

    Institute of Scientific and Technical Information of China (English)

    贾晓琳; 王东澍; 高志奇; 冯尔玲; 郑继平; 王恒樑; 郭桂英; 刘先凯

    2015-01-01

    炭疽芽胞杆菌(Bacillus anthracis)、蜡样芽胞杆菌(B. cereus)和苏云金芽胞杆菌(B. thuringiensis)均属于蜡样芽胞杆菌群,在遗传学上有很高的相似性.PlcR (Phospholipase C regulator)在蜡样芽胞杆菌中是十分重要的调控因子,但plcR基因在炭疽芽胞杆菌中发生一个无义突变导致在炭疽芽胞杆菌中产生一个截短PlcR蛋白.为了研究plcR基因对炭疽芽胞杆菌功能的影响,文章以蜡样芽胞杆菌CMCC6330基因组为模板,构建重组表达质粒pBE2A-plcR后导入炭疽芽胞杆菌疫苗株 A16R中获得重组菌株,对其进行表型分析.结果显示,炭疽芽胞杆菌重组菌株的溶血活性基本没有恢复,但恢复了部分神经鞘磷脂酶活性,表明将蜡样芽胞杆菌的 plcR基因导入炭疽芽胞杆菌后,可以直接激活神经鞘磷脂酶活性.%Bacillus anthracis,B. thuringiensis andB. cereus are members of theB. cereusgroup. They share high genetic similarity. Whereas plcR (Phospholipase C regulator) usually encodes a functional pleiotropic activator pro-tein inB. cereus andB. thuringiensis isolates, a characteristic nonsense mutation is found in allB. anthracisstrains investigated, making the gene dysfunctional. To study the function of PlcR inB. anthracis, we used theB. cereus CMCC63301genome as a template and constructed a recombinant expression plasmid pBE2A-plcR, and introduced it intothe B. anthracis vaccine strain A16R, and then analyzed the activity of the hemolysin and sphingomyelinase. The results showed that transformation ofB. anthracis with plasmid pBE2A-plcR carrying the nativeB. cereus plcR gene active the expression of sphingomyelinase gene, but did not activate expression of hemolysin genes ofB. anthracis A16R.

  15. Detection of Bacillus Anthracis Using Fluorescence Immunoassay with Quantum Dots Labels%荧光量子点免疫标记法检测炭疽芽孢杆菌

    Institute of Scientific and Technical Information of China (English)

    刘晓红; 罗金平; 田青; 刘春秀; 蔡新霞

    2011-01-01

    A method for detecting Bacillus anthracis (B. anthracis) was constructed with indirect immunofluorescence technique using quantum dots (QDs) as fluorescent probes. B. anthracis could be labeled with QDs selectively, that was proved by fluorescence microscope and F4500 spectrofluorometer. The laboratory-made system including biosensor and device was utilized as detection apparatus. A linear relationship of the fluorescence intensity and log total count of B. anthracis was obtained with correlation coefficient of 0. 9554 from 100 CFU/mL to 1 × 106 CFU/mL, and a low relative standard deviation of 2. 2%. By comparing the fluorescence intensity of B. anthracis and other bacillus, this method showed good specificity. Because of the simple operation, the detecting time is less (1 h) than traditional methods as well as quantitative detection, this method has potential prospect in monitoring B. anthracis and other biowarfare agents.%建立了荧光量子点标记-免疫分析技术联用检测炭疽芽孢杆菌的方法.通过抗原抗体反应,结合生物素与亲和素间的特异性相互作用,将QDs特异性标记在炭疽芽孢杆菌上,并利用荧光显微镜和荧光分光光度计进行了验证.采用实验室自制的便携式荧光检测系统对标记QDs的炭疽芽孢杆菌样品进行定量检测.结果表明,在炭疽芽孢杆菌浓度在100~1×106CFU/mL范围内,相对荧光强度与炭疽芽孢杆菌浓度呈良好的线性关系,相关系数R=0.9554,检测相对标准偏差为2.2%.通过与同菌属其它杆菌对比,证明本方法特异性良好.与传统方法相比,本方法操作简单,检测时间短(1 h),且能实现定量检测,在公共安全等方面有广泛的应用前景.

  16. Ecological economic benefit in sustainable development

    Institute of Scientific and Technical Information of China (English)

    Jiang Xuemin; Ren Long

    2006-01-01

    From the concept of ecological economic benefit, I put forward the general formula for the benefit of ecological economy and the appraisal methods of the ecological economy, Theory on ecological benefit and economic benefit is the base of the benefits of ecological economy To some extent, the development of ecological economy, theory and practice on eco-agriculture are both the production made from opposition and unify of ecological benefit and economic benefit. This paper discusses the "T" type structure, which will give the theoretical bases for enhancing the ecological and economical benefits.

  17. 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.

  18. The Bacillus anthracis arylamine N-acetyltransferase ((BACAN)NAT1) that inactivates sulfamethoxazole, reveals unusual structural features compared with the other NAT isoenzymes.

    Science.gov (United States)

    Pluvinage, Benjamin; Li de la Sierra-Gallay, Inés; Kubiak, Xavier; Xu, Ximing; Dairou, Julien; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2011-12-15

    Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that biotransform arylamine drugs. The Bacillus anthracis (BACAN)NAT1 enzyme affords increased resistance to the antibiotic sulfamethoxazole through its acetylation. We report the structure of (BACAN)NAT1. Unexpectedly, endogenous coenzymeA was present in the active site. The structure suggests that, contrary to the other prokaryotic NATs, (BACAN)NAT1 possesses a 14-residue insertion equivalent to the "mammalian insertion", a structural feature considered unique to mammalian NATs. Moreover, (BACAN)NAT1 structure shows marked differences in the mode of binding and location of coenzymeA when compared to the other NATs. This suggests that the mechanisms of cofactor recognition by NATs is more diverse than expected and supports the cofactor-binding site as being a unique subsite to target in drug design against bacterial NATs.

  19. 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.

  20. 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.