WorldWideScience

Sample records for nonproteolytic clostridium botulinum

  1. Growth of and toxin production by nonproteolytic Clostridium botulinum in cooked puréed vegetables at refrigeration temperatures.

    OpenAIRE

    Carlin, F; Peck, M W

    1996-01-01

    Seven strains of nonproteolytic Clostridium botulinum (types B, E, and F) were each inoculated into a range of anaerobic cooked puréed vegetables. After incubation at 10 degrees C for 15 to 60 days, all seven strains formed toxin in mushrooms, five did so in broccoli, four did so in cauliflower, three did so in asparagus, and one did so in kale. Growth kinetics of nonproteolytic C. botulinum type B in cooked mushrooms, cauliflower, and potatoes were determined at 16, 10, 8, and 5 degrees C. G...

  2. Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement.

    Science.gov (United States)

    Clauwers, Charlien; Vanoirbeek, Kristof; Delbrassinne, Laurence; Michiels, Chris W

    2016-05-15

    Group II nonproteolytic Clostridium botulinum (gIICb) strains are an important concern for the safety of minimally processed ready-to-eat foods, because they can grow and produce botulinum neurotoxin during refrigerated storage. The principles of control of gIICb by conventional food processing and preservation methods have been well investigated and translated into guidelines for the food industry; in contrast, the effectiveness of emerging processing and preservation techniques has been poorly documented. The reason is that experimental studies with C. botulinum are cumbersome because of biosafety and biosecurity concerns. In the present work, we report the construction of two nontoxigenic derivatives of the type E gIICb strain NCTC 11219. In the first strain, the botulinum toxin gene (bont/E) was insertionally inactivated with a retargeted intron using the ClosTron system. In the second strain, bont/E was exchanged for an erythromycin resistance gene using a new gene replacement strategy that makes use of pyrE as a bidirectional selection marker. Growth under optimal and stressed conditions, sporulation efficiency, and spore heat resistance of the mutants were unaltered, except for small differences in spore heat resistance at 70°C and in growth at 2.3% NaCl. The mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with gIICb. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in gIICb and other clostridia. The nontoxigenic mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with psychrotrophic Clostridium botulinum In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in clostridia. Copyright © 2016, American Society for

  3. Characterisation of non-toxigenic Clostridium spp. strains, to use as surrogates for non-proteolytic Clostridium botulinum in chilled food challenge testing.

    Science.gov (United States)

    Parker, M D; Barrett, P I; Shepherd, J; Price, L J; Bull, S D

    2015-01-01

    Under many of the conditions studied, a two-strain cocktail of non-toxigenic Clostridium spp. was found to be suitable as a surrogate for non-proteolytic Clostridium botulinum, and has the potential for use in chilled food challenge tests measuring growth. Non-toxigenic surrogates could also be used in thermal process screening studies. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Inability of non-proteolytic Clostridium botulinum to grow in mussels inoculated via immersion and packaged in high oxygen atmospheres.

    Science.gov (United States)

    Newell, Carter R; Doyle, Michael; Ma, Li

    2015-04-01

    A series of botulism challenge studies were conducted to determine if botulinum toxin would be produced in mussels (Mytilus edulis) inoculated with non-proteolytic Clostridium botulinum spores and held under modified atmosphere (MA) packaging conditions at normal (4 °C) and abusive (12 °C) temperatures. Spore mixtures of six strains of non-proteolytic C. botulinum were introduced into live mussels through immersion in a seawater solution with cultured algae. Mussels were packed in a commercial high-oxygen (60-65% O2) MA-package with a buffer, and also packed under a vacuum. Feeding live mussels cultured algae (10(4) cells/ml) with a C. botulinum spore suspension (10(3) spores/ml) in seawater at 4 °C for 6 h resulted in the uptake of spores into mussel tissue (500/g) and the mussel GI tract (100/g). Under all of the experimental conditions evaluated, none of the fresh mussels became toxic, even after spoilage and in the absence of oxygen. However, control samples using tuna or cooked mussel meats became toxic in the absence of oxygen. Botulinum toxin was not produced in fresh mussels packaged under the MA-packaging conditions evaluated, even at an abusive storage temperature (12 °C) for at least 12 days or at normal storage temperate (4 °C) for at least 21 days, which is beyond their shelf life. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Effect of heat treatment on survival of, and growth from, spores of nonproteolytic Clostridium botulinum at refrigeration temperatures.

    OpenAIRE

    Peck, M W; Lund, B M; Fairbairn, D A; Kaspersson, A S; Undeland, P C

    1995-01-01

    Spores of five type B, five type E, and two type F strains of nonproteolytic Clostridium botulinum were inoculated into tubes of an anaerobic meat medium plus lysozyme to give approximately 10(6) spores per tube. Sets of tubes were then subjected to a heat treatment, cooled, and incubated at 6, 8, 10, 12, and 25 degrees C for up to 60 days. Treatments equivalent to heating at 65 degrees C for 364 min, 70 degrees C for 8 min, and 75 degrees C for 27 min had little effect on growth and toxin fo...

  6. Detection limit of Clostridium botulinum spores in dried mushroom samples sourced from China.

    Science.gov (United States)

    Malakar, Pradeep K; Plowman, June; Aldus, Clare F; Xing, Zengtao; Zhao, Yong; Peck, Michael W

    2013-08-16

    A survey of dried mushrooms (Lentinula edodes (Shiitake) and Auricularia auricula (Wood Ear)) sourced from China was carried out to determine the natural contamination of these mushrooms with spores of proteolytic Clostridium botulinum and non-proteolytic C. botulinum. The mushrooms were collected from supermarkets and retailers in 21 cities in China during October 2008. Spore loads of C. botulinum in mushrooms have a degree of uncertainty and variability and this study contributes valuable data for determining prevalence of spores of C. botulinum in mushrooms. An optimized detection protocol that combined selective enrichment culture with multiplex PCR was used to test for spores of proteolytic and non-proteolytic C. botulinum. Detection limits were calculated, using a maximum likelihood protocol, from mushroom samples inoculated with defined numbers of spores of proteolytic C. botulinum or non-proteolytic C. botulinum. Based on the maximum likelihood detection limit, it is estimated that dried mushroom A. auricula contained botulinum, and botulinum. Dried L. edodes contained botulinum and it was not possible to determine reliable detection limits for spores of non-proteolytic C. botulinum using the current detection protocol. © 2013 Elsevier B.V. All rights reserved.

  7. Genetic diversity of the flagellin genes of Clostridium botulinum groups I and II.

    Science.gov (United States)

    Woudstra, Cedric; Lambert, Dominic; Anniballi, Fabrizio; De Medici, Dario; Austin, John; Fach, Patrick

    2013-07-01

    Botulinum neurotoxins (BoNTs) are produced by phenotypically and genetically different Clostridium species, including Clostridium botulinum and some strains of Clostridium baratii (serotype F) and Clostridium butyricum (serotype E). BoNT-producing clostridia responsible for human botulism encompass strains of group I (secreting proteases, producing toxin serotype A, B, or F, and growing optimally at 37°C) and group II (nonproteolytic, producing toxin serotype E, B, or F, and growing optimally at 30°C). Here we report the development of real-time PCR assays for genotyping C. botulinum strains of groups I and II based on flaVR (variable region sequence of flaA) sequences and the flaB gene. Real-time PCR typing of regions flaVR1 to flaVR10 and flaB was optimized and validated with 62 historical and Canadian C. botulinum strains that had been previously typed. Analysis of 210 isolates of European origin allowed the identification of four new C. botulinum flaVR types (flaVR11 to flaVR14) and one new flaVR type specific to C. butyricum type E (flaVR15). The genetic diversity of the flaVR among C. botulinum strains investigated in the present study reveals the clustering of flaVR types into 5 major subgroups. Subgroups 1, 3, and 4 contain proteolytic Clostridium botulinum, subgroup 2 is made up of nonproteolytic C. botulinum only, and subgroup 5 is specific to C. butyricum type E. The genetic variability of the flagellin genes carried by C. botulinum and the possible association of flaVR types with certain geographical areas make gene profiling of flaVR and flaB promising in molecular surveillance and epidemiology of C. botulinum.

  8. Genomes, neurotoxins and biology of Clostridium botulinum Group I and Group II.

    Science.gov (United States)

    Carter, Andrew T; Peck, Michael W

    2015-05-01

    Recent developments in whole genome sequencing have made a substantial contribution to understanding the genomes, neurotoxins and biology of Clostridium botulinum Group I (proteolytic C. botulinum) and C. botulinum Group II (non-proteolytic C. botulinum). Two different approaches are used to study genomics in these bacteria; comparative whole genome microarrays and direct comparison of complete genome DNA sequences. The properties of the different types of neurotoxin formed, and different neurotoxin gene clusters found in C. botulinum Groups I and II are explored. Specific examples of botulinum neurotoxin genes are chosen for an in-depth discussion of neurotoxin gene evolution. The most recent cases of foodborne botulism are summarised. Copyright © 2014 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

  9. Regulation of toxin synthesis in Clostridium botulinum and Clostridium tetani.

    Science.gov (United States)

    Connan, Chloé; Denève, Cécile; Mazuet, Christelle; Popoff, Michel R

    2013-12-01

    Botulinum and tetanus neurotoxins are structurally and functionally related proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin (BoNT) associates with non-toxic proteins (ANTPs) to form complexes of various sizes, whereas tetanus toxin (TeNT) does not form any complex. The BoNT and ANTP genes are clustered in a DNA segment called the botulinum locus, which has different genomic localization (chromosome, plasmid, phage) in the various Clostridium botulinum types and subtypes. The botulinum locus genes are organized in two polycistronic operons (ntnh-bont and ha/orfX operons) transcribed in opposite orientations. A gene called botR lying between the two operons in C. botulinum type A encodes an alternative sigma factor which regulates positively the synthesis of BoNT and ANTPs at the late exponential growth phase and beginning of the stationary phase. In Clostridium tetani, the gene located immediately upstream of tent encodes a positive regulatory protein, TetR, which is related to BotR. C. botulinum and C. tetani genomes contain several two-component systems and predicted regulatory orphan genes. In C. botulinum type A, four two-component systems have been found that positively or negatively regulate the synthesis of BoNT and ANTPs independently of BotR/A. The synthesis of neurotoxin in Clostridia seems to be under the control of complex network of regulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Characterization of Clostridium sp. RKD producing botulinum-like neurotoxin.

    Science.gov (United States)

    Dixit, Aparna; Dhaked, Ram Kumar; Alam, Syed Imteyaz; Singh, Lokendra

    2005-07-01

    A Gram positive, motile, rod-shaped, strictly anaerobic bacterium isolated from intestine of decaying fish was identified as Clostridium sp. RKD and produced a botulinum type B-like neurotoxin as suggested by mouse bioassay and protection with anti botulinum antibodies. The neurotoxicity was functionally characterized by the phrenic nerve hemi-diaphragm assay. Phylogenetic analysis based on 16S rDNA sequence, placed it at a different position from the reported strains of Clostridium botulinum. The strain exhibited differences from both Clostridium botulinum and Clostridium tetani with respect to morphological, biochemical and chemotaxonomic characteristics. Botulinum group specific and serotype specific primers amplified the DNA fragments of 260 and 727 bp, respectively, indicating presence of botulinum type 'B' toxin gene. Sequence of nearly 700 bp amplified using primers specific for botulinum neurotoxin type B gene, did not show any significant match in the database when subjected to BLAST search.

  11. 9 CFR 113.110 - Clostridium Botulinum Type C Bacterin-Toxoid.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Clostridium Botulinum Type C Bacterin... REQUIREMENTS Inactivated Bacterial Products § 113.110 Clostridium Botulinum Type C Bacterin-Toxoid. Clostridium Botulinum Type C Bacterin-Toxoid shall be produced from a culture of Clostridium botulinum Type C which has...

  12. Recombination and Insertion Events Involving the Botulinum Neurotoxin Complex Genes in Clostridium botulinum Types A, B, E and F and Clostridium butyricum Type E Strains

    Science.gov (United States)

    2009-10-05

    Clostridium botulinum types A, B, E and F and Clostridium butyricum type E strains Karen K Hill*1, Gary Xie2, Brian T Foley3, Theresa J Smith4, Amy C Munk2...ornl.gov; John C Detter - cdetter@lanl.gov * Corresponding author Abstract Background: Clostridium botulinum is a taxonomic designation for at least... botulinum neurotoxin complex genes in Clostridium botulinum types A, B, E and F and Clostridium butyricum type E strains. BMC Genomics 7:1-18 5a

  13. Arrangement of the Clostridium baratii F7 toxin gene cluster with identification of a σ factor that recognizes the botulinum toxin gene cluster promoters.

    Science.gov (United States)

    Dover, Nir; Barash, Jason R; Burke, Julianne N; Hill, Karen K; Detter, John C; Arnon, Stephen S

    2014-01-01

    Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. We sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. This TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.

  14. Molecular diversity of Clostridium botulinum and phenotypically similar strains.

    Science.gov (United States)

    Grenda, T; Kukier, E; Sieradzki, Z; Goldsztejn, M; Kwiatek, K

    2016-12-01

    This study was undertaken to examine phenotypic and genetic features of strains preliminary classified as Clostridium botulinum species. The phenotypic characteristics were assessed with different culture media and biochemical tests. The genetic characterization included detection of botulinum toxin genes by PCR and macrorestriction analysis with SmaI, XhoI and SacII by PFGE (Pulsed-field Gel Electrophoresis). Despite similar biochemical properties of all analysed strains, only 47% of them contained genes determining toxicity specific to C. botulinum species. The most valuable differentiation of C. botulinum and C. botulinum-like strains was obtained after SmaI digestion. The highest affinity was observed among C. botulinum type B profiles which was even up to 100%. It was found 100% of affinity between C. botulinum and C. botulinum-like strains, however, the similarity among C. botulinum and C. botulinum-like was generally lower than 80%.

  15. High pressure thermal inactivation of Clostridium botulinum type E endospores – kinetic modeling and mechanistic insights

    Directory of Open Access Journals (Sweden)

    Christian Andreas Lenz

    2015-07-01

    Full Text Available Cold-tolerant, neurotoxigenic, endospore forming Clostridium (C. botulinum type E belongs to the non-proteolytic physiological C. botulinum group II, is primarily associated with aquatic environments, and presents a safety risk for seafood. High pressure thermal (HPT processing exploiting the synergistic effect of pressure and temperature can be used to inactivate bacterial endospores.We investigated the inactivation of C. botulinum type E spores by (near isothermal HPT treatments at 300 – 1200 MPa at 30 – 75 °C for 1 s – 10 min. The occurrence of heat and lysozyme susceptible spore fractions after such treatments was determined. The experimental data were modeled to obtain kinetic parameters and represented graphically by isoeffect lines. In contrast to findings for spores of other species and within the range of treatment parameters applied, zones of spore stabilization (lower inactivation than heat treatments alone, large heat susceptible (HPT-induced germinated or lysozyme-dependently germinable (damaged coat layer spore fractions were not detected. Inactivation followed 1st order kinetics. DPA release kinetics allowed for insights into possible inactivation mechanisms suggesting a (poorly effective physiologic-like (similar to nutrient-induced germination at ≤ 450 MPa/≤ 45 °C and non-physiological germination at >500 MPa/>60 – 70 °C.Results of this study support the existence of some commonalities in the HPT inactivation mechanism of C. botulinum type E spores and Bacillus spores although both organisms have significantly different HPT resistance properties. The information presented here contributes to closing the gap in knowledge regarding the HPT inactivation of spore formers relevant to food safety and may help industrial implementation of HPT processing. The markedly lower HPT resistance of C. botulinum type E spores than spores from other C. botulinum types, could allow for the implementation of milder processes without

  16. Investigation of Clostridium botulinum group III's mobilome content

    NARCIS (Netherlands)

    Woudstra, Cédric; Maréchal, Le Caroline; Souillard, Rozenn; Anniballi, Fabrizio; Auricchio, Bruna; Bano, Luca; Bayon-Auboyer, Marie Hélène; Koene, Miriam; Mermoud, Isabelle; Brito, Roseane B.; Lobato, Francisco C.F.; Silva, Rodrigo O.S.; Dorner, Martin B.; Fach, Patrick

    2018-01-01

    Clostridium botulinum group III is mainly responsible for botulism in animals. It could lead to high animal mortality rates and, therefore, represents a major environmental and economic concern. Strains of this group harbor the botulinum toxin locus on an unstable bacteriophage. Since the release of

  17. Plasmidome Interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum Converts Strains of Independent Lineages into Distinctly Different Pathogens

    OpenAIRE

    Skarin, Hanna; Segerman, Bo

    2014-01-01

    Clostridium botulinum (group III), Clostridium novyi and Clostridium haemolyticum are well-known pathogens causing animal botulism, gas gangrene/black disease, and bacillary hemoglobinuria, respectively. A close genetic relationship exists between the species, which has resulted in the collective term C. novyi sensu lato. The pathogenic traits in these species, e.g., the botulinum neurotoxin and the novyi alpha toxin, are mainly linked to a large plasmidome consisting of plasmids and circular...

  18. Taxonomic relationships among Clostridium novyi Types A and B, Clostridium haemolyticum and Clostridium botulinum type C.

    Science.gov (United States)

    Nakamura, S; Kimura, I; Yamakawa, K; Nishida, S

    1983-05-01

    The present study was undertaken to examine the genetic relationships among the closely related species, Clostridium novyi types A and B, C. haemolyticum and C. botulinum type C. These species were tested for DNA-DNA homology and thermostability of DNA duplexes and sorted into three genetically related groups: I, C. novyi type A; II, C. novyi type B, C. haemolyticum and one C. botulinum type C strain (Stockholm); III, the remaining C. botulinum type C strains. A few biochemical criteria corresponding to the genetic differences were recommended to differentiate each group. These studies imply that C. haemolyticum might be considered as C. novyi type D and that there are two genetically different groups in C. botulinum type C.

  19. Structural Studies on Intact Clostridium botulinum Neurotoxins Complexed with Inhibitors Leading to Drug Design

    Science.gov (United States)

    2009-02-01

    TITLE:Structural Studies on Intact Clostridium botulinum Neurotoxins Complexed with Inhibitors Leading to Drug Design PRINCIPAL INVESTIGATOR: Dr...JAN 2009 4. TITLE AND SUBTITLE Structural Studies on Intact Clostridium botulinum 5a. CONTRACT NUMBER Neurotoxins Complexed with Inhibitors... Clostridium , botulinum , neurotoxin, zinc chelators, inhibitors, macromolecular crystallography, 3D structure 16. SECURITY CLASSIFICATION OF: 17

  20. Genomics of Clostridium botulinum group III strains.

    Science.gov (United States)

    Sakaguchi, Yoshihiko; Suzuki, Tomonori; Yamamoto, Yumiko; Nishikawa, Atsushi; Oguma, Keiji

    2015-05-01

    In Clostridium botulinum, the characteristics of type C and D strains are quite different from other types, and they are classified as group III. They produce C2 binary toxin and C3 exoenzyme in addition to type C and D neurotoxins. Two different phages and many plasmids are identified in the organisms. The genes of neurotoxin and C3 exoenzyme are converted from toxigenic strains to non-toxigenic strains by the specific bacteriophages (phages), whereas, the C2 toxin gene is carried by large or small plasmids. Classification of type C and D strains has been in confusion because 1) antigenicity of type C and D neurotoxins is complex, 2) the cells produce two types of toxins, neurotoxin and C2 toxin, and 3) some non-toxigenic strains can be converted to produce C or D neurotoxin by the infection with phages. Until now, entire nucleotide sequences of cell chromosomes, phages, and plasmids have been determined. Since both genetic and protein-chemical analyses have been clarifying the above confusions, these data are reviewed historically. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  1. Genomic organization and diversity of Clostridium botulinum group III

    OpenAIRE

    Skarin, Hanna

    2015-01-01

    Botulism is caused by botulinum neurotoxins (BoNTs) produced by the spore forming strictly anaerobic bacterium Clostridium botulinum. Seven different types of BoNTs (type A-F) have so far been established on the basis of neutralization with different antibodies. Botulism affects both humans and animals, and there are occasionally large-scale outbreaks of high mortality in animals. Especially large outbreaks of avian botulism have been reported from various countries, including Sweden. Other a...

  2. A study on the toxigenesis by Clostridium botulinum in nitrate and nitrite-reduced dry fermented sausages.

    Science.gov (United States)

    Hospital, Xavier F; Hierro, Eva; Stringer, Sandra; Fernández, Manuela

    2016-02-02

    Nitrite has been traditionally used to control Clostridium botulinum in cured meat products. However, in the case of dry fermented sausages, environmental factors such as pH, aw and the competitive microbiota may exert a more relevant role than nitrite in the inhibition of the growth and toxin production by C. botulinum. In this challenge test study, two varieties of Mediterranean dry sausages (salchichón and fuet) were inoculated with spores of C. botulinum Group I (proteolytic) and C. botulinum Group II (nonproteolytic). Sausages were prepared with 150 mg/kg of NaNO3 and 150 mg/kg of NaNO2 (maximum ingoing amounts allowed by the European Union regulation), with a 25% and 50% reduction, and without nitrate/nitrite. The initial pH in both products was 5.6, and decreased to values below 5.0 in salchichón and to 5.2 in fuet. Lactic acid bacteria counts reached 8-9 log cfu/g after fermentation. The aw decreased from initial values of 0.96 to about 0.88-0.90 at the end of ripening. Botulinum neurotoxin was not detected in any of the sausages, including those manufactured without nitrate and nitrite. Despite the environmental conditions were within the range for germination and growth of C. botulinum Group I during the first 8 days of the ripening process in fuet and 10-12 days in salchichón, acidity, aw and incubation temperature combined to inhibit the production of toxin, independently of the concentration of curing agents. Although decreasing or even removing nitrate/nitrite from the formula did not compromise safety regarding C. botulinum in the conditions tested in this study, their antimicrobial role should not be underestimated in the case that other hurdles could fail or other ripening conditions were used, and also considering the effect of nitrite on other pathogens.

  3. Effect of sporulation medium and its divalent cation content on the heat and high pressure resistance of Clostridium botulinum type E spores.

    Science.gov (United States)

    Lenz, Christian A; Vogel, Rudi F

    2014-12-01

    Clostridium (C.) botulinum type E belongs to the non-proteolytic physiological C. botulinum group II and produces the highly potent Botulinum neurotoxin E (BoNT/E) even at refrigerated temperatures. As C. botulinum type E spores are highly prevalent in aquatic environments, seafood and fishery products are commonly associated with this organism. Hydrostatic high pressure (HHP) treatments, or treatments combining HHP with elevated temperatures (HHPT), can be used to improve traditional preservation methods and increase food safety, quality and durability. In this study, we assessed the effect of different sporulation media and cation concentration on the heat resistance, HHP resistance, and HHPT resistance of spores from three C. botulinum type E strains. SFE (sediment fish extract) sporulation media yielded the most resistant spores, whereas, in M140 media, the least resistant spores were produced. Furthermore our results indicate that the divalent cation content (Ca(2+), Mg(2+) and Mn(2+)) plays a role in the differential development of C. botulinum type E spore resistance to heat, HHP and HHPT in different media. Calcium cations confer heat and HPPT resistance to spores, while high amounts of magnesium cations appear to have a negative effect. Manganese cations in low concentrations are important for the development resistance to HPP and HPPT treatments, but not heat alone. This study provides valuable information on the nature of non-proteolytic C. botulinum type E spores grown in different media. The data provided here can be useful to the food industry and to researchers when considering spore properties in food safety risk assessment and the experimental design of future inactivation studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Organization and regulation of the neurotoxin genes in Clostridium botulinum and Clostridium tetani.

    Science.gov (United States)

    Raffestin, Stéphanie; Marvaud, Jean Christophe; Cerrato, Rosario; Dupuy, Bruno; Popoff, Michel R

    2004-04-01

    Botulinum and tetanus neurotoxins are structurally and functionally related 150 kDa proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin associates with non-toxic proteins to form complexes of various sizes. The botulinum neurotoxin and non-toxic protein genes are clustered in a DNA segment called the botulinum locus. This locus is probably located on a mobile or degenerate mobile element, which accounts for the various genomic localizations (chromosome, plasmid, phage) in different Clostridium botulinum types. The botulinum neurotoxin and non-toxic protein genes are organized in two polycistronic operons (ntnh-bont and ha operons) transcribed in opposite orientations. The gene that separates the two operons of the botulinum locus in C. botulinum A encodes a 21 kDa protein BotR/A, which is a positive regulator of the expression of the botulinum locus genes. Similarly, in Clostridium tetani, the gene located immediately upstream of the tetanus toxin gene, encodes a positive regulatory protein, TetR. BotR and TetR are possibly alternative sigma factors related to TxeR and UviA, which regulate C. difficile toxin and C. perfringens bacteriocin production, respectively. TxeR and UviA define a new sub-group of the sigma(70) family of RNA polymerase initiation factors. In addition, the C. botulinum genome contains predicted two-component system genes, some of which are possibly involved in regulation of toxinogenesis.

  5. Historical and current perspectives on Clostridium botulinum diversity.

    Science.gov (United States)

    Smith, Theresa J; Hill, Karen K; Raphael, Brian H

    2015-05-01

    For nearly one hundred years, researchers have attempted to categorize botulinum neurotoxin-producing clostridia and the toxins that they produce according to biochemical characterizations, serological comparisons, and genetic analyses. Throughout this period the bacteria and their toxins have defied such attempts at categorization. Below is a description of both historic and current Clostridium botulinum strain and neurotoxin information that illustrates how each new finding has significantly added to the knowledge of the botulinum neurotoxin-containing clostridia and their diversity. Published by Elsevier Masson SAS.

  6. Genomic characterization of Italian Clostridium botulinum group I strains.

    Science.gov (United States)

    Giordani, Francesco; Fillo, Silvia; Anselmo, Anna; Palozzi, Anna Maria; Fortunato, Antonella; Gentile, Bernardina; Azarnia Tehran, Domenico; Ciammaruconi, Andrea; Spagnolo, Ferdinando; Pittiglio, Valentina; Anniballi, Fabrizio; Auricchio, Bruna; De Medici, Dario; Lista, Florigio

    2015-12-01

    Clostridium botulinum is a gram-positive bacterium capable of producing the botulinum neurotoxin, a powerful poison that causes botulism, a severe neuroparalytic disease. Its genome has been sequenced entirely and its gene content has been analyzed. To date, 19 full genomes and 64 draft genomes are available. The geographical origin of these genomes is predominantly from the US. In the present study, 10 Italian genomes of C. botulinum group I were analyzed and compared with previously sequenced group I genomes, in order to genetically characterize the Italian population of C. botulinum group I and to investigate the phylogenetic relationships among different lineages. Using the suites of software ClonalFrame and ClonalOrigin to perform genomic analysis, we demonstrated that Italian C. botulinum group I population is phylogenetically heterogeneous encompassing different and distant lineages including overseas strains, too. Moreover, a high recombination rate was demonstrated in the evolution of C. botulinum group I species. Finally, genome sequencing of the strain 357 led us to identify a novel botulinum neurotoxin subtype, F8. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Identification and Biochemical Characterization of Small-Molecule Inhibitors of Clostridium Botulinum Neurotoxin Serotype A

    Science.gov (United States)

    2009-08-01

    inhibitors of Clostridium botulinum neurotoxin serotype A (BoNT/A). Virtual screening was initially performed by computationally docking com- pounds of the...species Clostridium botulinum , C. baratii, and C. butyricum, consist of seven immunologically distinct serotypes (A to G). BoNTs are synthesized as 150...4. TITLE AND SUBTITLE Identification and biochemical characterization of small-molecule inhibitors of Clostridium botulinum neurotoxin serotype

  8. Structural Studies on Intact Clostridium Botulinum Neurotoxins Complexed with Inhibitors Leading to Drug Design

    Science.gov (United States)

    2006-02-01

    structure1. Introduction Tetanus neurotoxin (TeNT) produced by Clostridium tetani and the seven antigenically distinct botulinum neurotoxins (BoNT/A-G...2-0011 TITLE: Structural Studies on Intact Clostridium Botulinum Neurotoxins Complexed with Inhibitors Leading to Drug...DATES COVERED (From - To) 28 Jan 2005 – 27 Jan 2006 4. TITLE AND SUBTITLE Structural Studies on Intact Clostridium Botulinum Neurotoxins Complexed

  9. An atypical Clostridium strain related to the Clostridium botulinum group III strain isolated from a human blood culture.

    Science.gov (United States)

    Bouvet, Philippe; Ruimy, Raymond; Bouchier, Christiane; Faucher, Nathalie; Mazuet, Christelle; Popoff, Michel R

    2014-01-01

    A nontoxigenic strain isolated from a fatal human case of bacterial sepsis was identified as a Clostridium strain from Clostridium botulinum group III, based on the phenotypic characters and 16S rRNA gene sequence, and was found to be related to the mosaic C. botulinum D/C strain according to a multilocus sequence analysis of 5 housekeeping genes.

  10. Investigation of Clostridium botulinum group III's mobilome content.

    Science.gov (United States)

    Woudstra, Cédric; Le Maréchal, Caroline; Souillard, Rozenn; Anniballi, Fabrizio; Auricchio, Bruna; Bano, Luca; Bayon-Auboyer, Marie-Hélène; Koene, Miriam; Mermoud, Isabelle; Brito, Roseane B; Lobato, Francisco C F; Silva, Rodrigo O S; Dorner, Martin B; Fach, Patrick

    2018-02-01

    Clostridium botulinum group III is mainly responsible for botulism in animals. It could lead to high animal mortality rates and, therefore, represents a major environmental and economic concern. Strains of this group harbor the botulinum toxin locus on an unstable bacteriophage. Since the release of the first complete C. botulinum group III genome sequence (strain BKT015925), strains have been found to contain others mobile elements encoding for toxin components. In this study, seven assays targeting toxin genes present on the genetic mobile elements of C. botulinum group III were developed with the objective to better characterize C. botulinum group III strains. The investigation of 110 C. botulinum group III strains and 519 naturally contaminated samples collected during botulism outbreaks in Europe showed alpha-toxin and C2-I/C2-II markers to be systematically associated with type C/D bont-positive samples, which may indicate an important role of these elements in the pathogenicity mechanisms. On the contrary, bont type D/C strains and the related positive samples appeared to contain almost none of the markers tested. Interestingly, 31 bont-negative samples collected on farms after a botulism outbreak revealed to be positive for some of the genetic mobile elements tested. This suggests loss of the bont phage, either in farm environment after the outbreak or during laboratory handling. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Detection of Clostridium botulinum in natural sweetening.

    Science.gov (United States)

    Nakano, H; Yoshikuni, Y; Hashimoto, H; Sakaguchi, G

    1992-06-01

    Various sugar products were examined for contamination with C. botulinum spores. Type A, B and C spores were detected in three of 56 samples of sugar for apiculture, which may attest the significance of bee-feed as a source of contamination of honey. The heavy contamination of honey with C. botulinum spores sometimes encountered, however, can not be explained unless some other factors, e.g., that allowing germination and multiplication of the spores somewhere during honey production, are found. Type A spores were detected in some samples of raw sugar and molasses and also in two of 41 samples of brown sugar lump, but not in refined sugar or other various samples taken at a sugar factory or in sugar cane left on the field in Okinawa. The fact that some natural sweetenings are contaminated with C. botulinum spores, even in low concentrations, may be food-hygienically important.

  12. Two-component systems and toxinogenesis regulation in Clostridium botulinum.

    Science.gov (United States)

    Connan, Chloé; Popoff, Michel R

    2015-05-01

    Botulinum neurotoxins (BoNTs) are the most potent toxins ever known. They are mostly produced by Clostridium botulinum but also by other clostridia. BoNTs associate with non-toxic proteins (ANTPs) to form complexes of various sizes. Toxin production is highly regulated through complex networks of regulatory systems involving an alternative sigma factor, BotR, and at least 6 recently described two-component systems (TCSs). TCSs allow bacteria to sense environmental changes and to respond to various stimuli by regulating the expression of specific genes at a transcriptional level. Several environmental stimuli have been identified to positively or negatively regulate toxin synthesis; however, the link between environmental stimuli and TCSs is still elusive. This review aims to highlight the role of TCSs as a central point in the regulation of toxin production in C. botulinum. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  13. CRYSTAL STRUCTURE OF CLOSTRIDIUM BOTULINUM NEUROTOXIN SEROTYPE B

    International Nuclear Information System (INIS)

    SWAMINATHAN, S.; ESWARAMOORTHY, S.

    2001-01-01

    The toxigenic strains of Clostridium botulinum produce seven serologically distinct types of neurotoxins labeled A - G (EC 3.4.24.69), while Clostridium tetani produces tetanus neurotoxin (EC 3.4.24.68). Botulinum and tetanus neurotoxins (BoNTs and TeNT) are produced as single inactive chains of molecular mass of approximately 150 kDa. Most of these neurotoxins are released after being cleaved into two chains, a heavy chain (HI) of 100 kDa and a light chain (L) of 50 kDa held together by an interchain disulfide bond, by tissue proteinases. BoNT/E is released as a single chain but cleaved by host proteinases[1]. Clostvidium botulinum neurotoxins are extremely poisonous proteins with their LD(sub 50) for humans in the range of 0.1 - 1 ng kg(sup -1)[2]. Botulinum neurotoxins are responsible for neuroparalytic syndromes of botulism characterized by serious neurological disorders and flaccid paralysis. BoNTs block the release of acetylcholine at the neuromuscular junction causing flaccid paralysis while TeNT blocks the release of neurotransmitters like glycine and(gamma)-aminobutyric acid (GABA) in the inhibitory interneurons of the spinal cord resulting in spastic paralysis. In spite of different clinical symptoms, their aetiological agents intoxicate neuronal cells in the same way and these toxins have similar structural organization[3

  14. CRYSTAL STRUCTURE OF CLOSTRIDIUM BOTULINUM NEUROTOXIN SEROTYPE B.

    Energy Technology Data Exchange (ETDEWEB)

    SWAMINATHAN,S.; ESWARAMOORTHY,S.

    2001-11-19

    The toxigenic strains of Clostridium botulinum produce seven serologically distinct types of neurotoxins labeled A - G (EC 3.4.24.69), while Clostridium tetani produces tetanus neurotoxin (EC 3.4.24.68). Botulinum and tetanus neurotoxins (BoNTs and TeNT) are produced as single inactive chains of molecular mass of approximately 150 kDa. Most of these neurotoxins are released after being cleaved into two chains, a heavy chain (HI) of 100 kDa and a light chain (L) of 50 kDa held together by an interchain disulfide bond, by tissue proteinases. BoNT/E is released as a single chain but cleaved by host proteinases [1]. Clostvidium botulinum neurotoxins are extremely poisonous proteins with their LD{sub 50} for humans in the range of 0.1 - 1 ng kg{sup -1} [2]. Botulinum neurotoxins are responsible for neuroparalytic syndromes of botulism characterized by serious neurological disorders and flaccid paralysis. BoNTs block the release of acetylcholine at the neuromuscular junction causing flaccid paralysis while TeNT blocks the release of neurotransmitters like glycine and {gamma}-aminobutyric acid (GABA) in the inhibitory interneurons of the spinal cord resulting in spastic paralysis. In spite of different clinical symptoms, their aetiological agents intoxicate neuronal cells in the same way and these toxins have similar structural organization [3].

  15. Botulinum neurotoxin homologs in non-Clostridium species.

    Science.gov (United States)

    Mansfield, Michael J; Adams, Jeremy B; Doxey, Andrew C

    2015-01-30

    Clostridial neurotoxins (CNTs) are the deadliest toxins known and the causative agents of botulism and tetanus. Despite their structural and functional complexity, no CNT homologs are currently known outside Clostridium. Here, we report the first homologs of Clostridium CNTs within the genome of the rice fermentation organism Weissella oryzae SG25. One gene in W. oryzae S25 encodes a protein with a four-domain architecture and HExxH protease motif common to botulinum neurotoxins (BoNTs). An adjacent gene with partial similarity to CNTs is also present, and both genes seem to have been laterally transferred into the W. oryzae genome from an unknown source. Identification of mobile, CNT-related genes outside of Clostridium has implications for our understanding of the evolution of this important toxin family. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. First report of an infant botulism case due to Clostridium botulinum type Af.

    Science.gov (United States)

    de Jong, Laura I T; Fernández, Rafael A; Pareja, Virtudes; Giaroli, Gabriel; Guidarelli, Sergio R; Dykes, Janet K; Lúquez, Carolina

    2015-02-01

    Most infant botulism cases worldwide are due to botulinum toxin types A and B. Rarely, Clostridium botulinum strains that produce two serotypes (Ab, Ba, and Bf) have also been isolated from infant botulism cases. This is the first reported case of infant botulism due to C. botulinum type Af worldwide. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins.

    Science.gov (United States)

    Barash, Jason R; Arnon, Stephen S

    2014-01-15

    Clostridium botulinum strain IBCA10-7060, isolated from a patient with infant botulism, produced botulinum neurotoxin type B (BoNT/B) and another BoNT that, by use of the standard mouse bioassay, could not be neutralized by any of the Centers for Disease Control and Prevention-provided monovalent polyclonal botulinum antitoxins raised against BoNT types A-G.  The combining of antitoxins to neutralize the toxicity of known bivalent C. botulinum strains Ab, Ba, Af, and Bf also failed to neutralize the second BoNT. Analysis of culture filtrate by double immunodiffusion yielded a single line of immunoprecipitate with anti-A, anti-B, and anti-F botulinum antitoxins but not with anti-E antitoxin. A heptavalent F(ab')2 botulinum antitoxin A-G obtained from the US Army also did not neutralize the second BoNT. An antitoxin raised against IBCA10-7060 toxoid protected mice against BoNT/B (Okra) and against the second BoNT but did not protect mice against BoNT/A (Hall) or BoNT/F (Langeland). The second BoNT thus fulfilled classic criteria for being designated BoNT/H. IBCA10-7060 is the first C. botulinum type Bh strain to be identified. BoNT/H is the first new botulinum toxin type to be recognized in >40 years, and its recognition could not have been accomplished without the availability of the mouse bioassay.

  18. Positive regulation of botulinum neurotoxin gene expression by CodY in Clostridium botulinum ATCC 3502.

    Science.gov (United States)

    Zhang, Zhen; Dahlsten, Elias; Korkeala, Hannu; Lindström, Miia

    2014-12-01

    Botulinum neurotoxin, produced mainly by the spore-forming bacterium Clostridium botulinum, is the most poisonous biological substance known. Here, we show that CodY, a global regulator conserved in low-G+C Gram-positive bacteria, positively regulates the botulinum neurotoxin gene expression. Inactivation of codY resulted in decreased expression of botA, encoding the neurotoxin, as well as in reduced neurotoxin synthesis. Complementation of the codY mutation in trans rescued neurotoxin synthesis, and overexpression of codY in trans caused elevated neurotoxin production. Recombinant CodY was found to bind to a 30-bp region containing the botA transcription start site, suggesting regulation of the neurotoxin gene transcription through direct interaction. GTP enhanced the binding affinity of CodY to the botA promoter, suggesting that CodY-dependent neurotoxin regulation is associated with nutritional status. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  19. Clostridium tepidum sp. nov., a close relative of Clostridium sporogenes and Clostridium botulinum Group I.

    Science.gov (United States)

    Dobritsa, Anatoly P; Kutumbaka, Kirthi K; Werner, Kirsten; Wiedmann, Martin; Asmus, Aaron; Samadpour, Mansour

    2017-07-01

    Obligately anaerobic, Gram-stain-positive, spore-forming bacteria indistinguishable by pulsed-field gel electrophoresis were isolated from non-dairy protein shakes in bloated bottles. One of the isolates, strain IEH 97212T, was selected for further study. The strain was closely related to Clostridium sporogenes and Clostridium botulinum Group 1 based on 16S rRNA gene sequence similarities. Phylogenetic analysis also showed that strain IEH 97212T and strain PE (=DSM 18688), a bacterium isolated from solfataric mud, had identical 16S rRNA gene sequences. Strains IEH 97 212T and DSM 18 688 were relatively more thermophilic (temperature range for growth: 30-55 °C) and less halotolerant [growth range: 0-2.5 % (w/v) NaCl] than C. sporogenes and C. botulinum. They were negative for catalase, oxidase, urease and l-pyrrolidonyl-arylamidase and did not produce indole. The strains produced acid from d-glucose, maltose and trehalose, and hydrolysed gelatin, but did not hydrolyse aesculin. The end-products of growth included acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, isocaproic acid, phenylpropionic acid, 2-piperidinone, 2-pyrrolidinone and gas(es). The predominant fatty acids were C14 : 0, C16 : 0 and C18 : 1ω9c. The genomic DNA G+C content of strains IEH 97212T and DSM 18688 was 26.9 and 26.7 mol%, respectively. According to the digital DNA-DNA hybridization data, the relatedness of these strains was 98.4 %, while they showed only 35.7-36.0 % relatedness to C. sporogenes. Based on the results of this polyphasic study, these strains represent a novel species, for which the name Clostridium tepidum sp. nov. is proposed, with the type strain IEH 97212T (=NRRL B-65463T=DSM 104389T).

  20. Physical Characterization of Clostridium Botulinum Neurotoxin Genes

    Science.gov (United States)

    1993-10-01

    modern food -preserving processes in Western countries has made 1 outbreaks of botulism extremely rare. The frequent use of C.botulinum as a test...organism in the food industry, and the growing use of the toxin by neurobiochemists, has, however, led to the development of human vaccines. The...390v 400v DOTE [• - F -TK--VQ -- 0Q ---YI G Q KM YLFKL NS N - S - N I G NF N K 383 DOTFIFT ED L AINIK FIKIV C RINIT YIF I K MCF LVPIN L L D DDIDII YIT

  1. Structures of engineered Clostridium botulinum neurotoxin derivatives

    International Nuclear Information System (INIS)

    Masuyer, Geoffrey; Stancombe, Patrick; Chaddock, John A.; Acharya, K. Ravi

    2011-01-01

    The crystal structures of engineered C. botulinum neurotoxin–SNARE derivatives have been and exhibit strong stability of the LHn fragment. Targeted secretion inhibitors (TSIs) are a new class of engineered biopharmaceutical molecules derived from the botulinum neurotoxins (BoNTs). They consist of the metalloprotease light chain (LC) and translocation domain (Hn) of BoNT; they thus lack the native toxicity towards motor neurons but are able to target soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) proteins. These functional fragment (LHn) derivatives are expressed as single-chain proteins and require post-translational activation into di-chain molecules for function. A range of BoNT derivatives have been produced to demonstrate the successful use of engineered SNARE substrate peptides at the LC–Hn interface that gives these molecules self-activating capabilities. Alternatively, recognition sites for specific exoproteases can be engineered to allow controlled activation. Here, the crystal structures of three LHn derivatives are reported between 2.7 and 3.0 Å resolution. Two of these molecules are derivatives of serotype A that contain a SNARE peptide. Additionally, a third structure corresponds to LHn serotype B that includes peptide linkers at the exoprotease activation site. In all three cases the added engineered segments could not be modelled owing to disorder. However, these structures highlight the strong interactions holding the LHn fold together despite the inclusion of significant polypeptide sequences at the LC–Hn interface

  2. Cellular Entry of Clostridium perfringens Iota-Toxin and Clostridium botulinum C2 Toxin

    Directory of Open Access Journals (Sweden)

    Masaya Takehara

    2017-08-01

    Full Text Available Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin are composed of two non-linked proteins, one being the enzymatic component and the other being the binding/translocation component. These latter components recognize specific receptors and oligomerize in plasma membrane lipid-rafts, mediating the uptake of the enzymatic component into the cytosol. Enzymatic components induce actin cytoskeleton disorganization through the ADP-ribosylation of actin and are responsible for cell rounding and death. This review focuses upon the recent advances in cellular internalization of clostridial binary toxins.

  3. Risk assessment of proteolytic Clostridium botulinum in canned foie gras.

    Science.gov (United States)

    Membré, Jeanne-Marie; Diao, Moctar; Thorin, Chantal; Cordier, Grégoire; Zuber, François; André, Stéphane

    2015-10-01

    In this study, a risk assessment of proteolytic Clostridium botulinum in canned foie gras was performed, the number of illnesses per year in France due to C. botulinum in foie gras was estimated. Data on initial level in raw materials were collected at manufacturers and analysed using a Negative Binomial distribution. The effect of the usual foie gras heat treatment (equivalent time at 121 °C: F0=0.5 min) was considered at two levels: first, it led to an inactivation (estimated to 2.3 log); second it led to a spore injury and then to a spore inhibition. This latter effect was assessed by analysing data from a challenge test study carried out with Clostridium sporogenes spores in the foie gras product. The probability of spore recovering after thermal inhibition was estimated to 9.5×10(-8) (corresponding to 7.0 log). The data on the consumption pattern were collected on the French market. The Quantitative Microbiological Risk Assessment (QMRA) model and all the assumptions are reported in detail in the study. The initial contamination of raw materials, effect of thermal treatment on microbial inactivation and spore inhibition were handled mathematically using a probabilistic framework, considering only the variability dimension. The model was implemented in Excel and run through Monte Carlo simulation, using @Risk software. In parallel, epidemiological data collected from the French Institute for Public Health Surveillance during the period 2001-2012 were used to estimate an Appropriate Level Of Protection (ALOP) and then a Food Safety Objective (FSO): ALOP equalled to 2.5×10(-3) illnesses per million inhabitant per year, FSO equalled to 1.4×10(-9) foie gras portions containing C. botulinum spore (expressed in decimal logarithm, FSO=-8.9). The QMRA model output values were smaller, but on the same order of magnitude as these two figures: 8.0×10(-4) illnesses per million inhabitants per year, and, 4.5×10(-10) (-9.3 log) foie gras portions containing C

  4. The Structure of the Neurotoxin- Associated Protein HA33/A from Clostridium botulinum Suggests a Reoccurring Beta-Trefoil Fold in the Progenitor Toxin Complex

    National Research Council Canada - National Science Library

    Arndt, Joseph W; Gu, Jenny; Jaroszewski, Lukasz; Schwarzenbacher, Robert; Hanson, Michael A; Lebeda, Frank L; Stevens, Raymond C

    2004-01-01

    The hemagglutinating protein HA33 from Clostridium botulinum is associated with the large botulinum neurotoxin secreted complexes and is critical in toxin protection, internalization, and possibly activation...

  5. Phenotypic and Genotypic Characterization of Bacteriocinogenic Enterococci Against Clostridium botulinum.

    Science.gov (United States)

    Shehata, Awad A; Tarabees, Reda; Basiouni, Shereen; Gamil, Mahmoud; Kamal, Ahmed S; Krüger, Monika

    2017-06-01

    The present study aimed to characterize Enterococcus faecalis (n = -6) and Enterococcus faecium (n = 1) isolated from healthy chickens to find a novel perspective probiotic candidate that antagonize Clostridium botulinum types A, B, D, and E. The isolated enterococci were characterized based on phenotypic properties, PCR, and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF). The virulence determinants including hemolytic activity on blood agar, gelatinase activity, sensitivity to vancomycin, and presence of gelatinase (gelE) and enterococcal surface protein (esp) virulence genes were investigated. Also, the presence of enterocin structural genes enterocin A, enterocin B, enterocin P, enterocin L50A/B, bacteriocin 31, enterocin AS48, enterocin 1071A/1071B, and enterocin 96 were assessed using PCR. Lastly, the antagonistic effect of the selected Enterococcus spp. on the growth of C. botulinum types A, B, D, and E was studied. The obtained results showed that four out of six E. faecalis and one E. faecium proved to be free from the tested virulence markers. All tested enterococci strains exhibited more than one of the tested enterocin. Interestingly, E. faecalis and E. faecium significantly restrained the growth of C. botulinum types A, B, D, and E. In conclusion, although, the data presented showed that bacteriocinogenic Enterococcus strains lacking of virulence determinants could be potentially used as a probiotic candidate against C. botulinum in vitro; however, further investigations are still urgently required to verify the beneficial effects of the tested Enterococcus spp. in vivo.

  6. Occurrence of human pathogenic Clostridium botulinum among healthy dairy animals: an emerging public health hazard.

    Science.gov (United States)

    Abdel-Moein, Khaled A; Hamza, Dalia A

    2016-01-01

    The current study was conducted to investigate the occurrence of human pathogenic Clostridium botulinum in the feces of dairy animals. Fecal samples were collected from 203 apparently healthy dairy animals (50 cattle, 50 buffaloes, 52 sheep, 51 goats). Samples were cultured to recover C. botulinum while human pathogenic C. botulinum strains were identified after screening of all C. botulinum isolates for the presence of genes that encode toxins type A, B, E, F. The overall prevalence of C. botulinum was 18.7% whereas human pathogenic C. botulinum strains (only type A) were isolated from six animals at the rates of 2, 2, 5.8, and 2% for cattle, buffaloes, sheep, and goats, respectively. High fecal carriage rates of C. botulinum among apparently healthy dairy animals especially type A alarm both veterinary and public health communities for a potential role which may be played by dairy animals in the epidemiology of such pathogen.

  7. Fate of Clostridium botulinum and incidence of pathogenic clostridia in biogas processes.

    Science.gov (United States)

    Fröschle, B; Messelhäusser, U; Höller, C; Lebuhn, M

    2015-10-01

    This study aimed to assess the sanitary situation in agricultural biogas plants (BP) regarding pathogenic Clostridium spp. The incidence of Clostridium botulinum, Clostridium difficile, Clostridium novyi, Clostridium haemolyticum, Clostridium septicum and Clostridium chauvoei was investigated in 154 plant and animal substrates, digester sludges and digestates from full-scale BP using a method combining microbial enrichment with Real-Time PCR. The investigated clostridia were absent in the samples, except for Cl. novyi that was barely present (3·9%) and Cl. difficile that was more frequently detected (44·8%). Clostridium botulinum exposed to lab-scale digesters in sentinel chambers was reduced with D-values of 34·6 ± 11·2 days at 38°C and 1·0 ± 0·2 days at 55°C. These findings indicate minor relevance of clostridial pathogens in BP and an improved sanitary quality of the digestion product compared to untreated substrates concerning Cl. botulinum. However, the frequent detection of Cl. difficile opens questions on the durability of this organism in manure digestion lines. This is the first study providing data on the reduction of Cl. botulinum during biogas processes that scientifically invalidate contrary claims by some media in the public. Furthermore, the results improve the fragmentary knowledge on the prevalence of several clostridial pathogens in agricultural biogas production. © 2015 The Society for Applied Microbiology.

  8. Genotyping Clostridium botulinum toxinotype A isolates from patients using amplified rDNA restriction analysis.

    Science.gov (United States)

    Pourshafie, M; Vahdani, P; Popoff, M

    2005-10-01

    In this study, the application of amplified rDNA restriction analysis (ARDRA) for characterizing Clostridium botulinum toxinotype A strains isolated from individuals with botulism was evaluated. Ten restriction enzymes were tested for their suitability in ARDRA as a typing method and HhaI was selected for the best outcome. Analysis of HhaI restriction profiles of the amplified products divided C. botulinum isolates into three clusters. Non-toxigenic Clostridium sporogenes strains showed an ARDRA restriction pattern that was distinct from those observed for C. botulinum. The successful use of ARDRA for subdivision of C. botulinum in this study confirmed that this technique is a powerful method for typing of C. botulinum toxinotype A clonal diversity. In addition, it is rapid, sensitive and simple.

  9. Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens.

    Science.gov (United States)

    Skarin, Hanna; Segerman, Bo

    2014-01-01

    Clostridium botulinum (group III), Clostridium novyi and Clostridium haemolyticum are well-known pathogens causing animal botulism, gas gangrene/black disease, and bacillary hemoglobinuria, respectively. A close genetic relationship exists between the species, which has resulted in the collective term C. novyi sensu lato. The pathogenic traits in these species, e.g., the botulinum neurotoxin and the novyi alpha toxin, are mainly linked to a large plasmidome consisting of plasmids and circular prophages. The plasmidome of C. novyi sensu lato has so far been poorly characterized. In this study we explored the genomic relationship of a wide range of strains of C. novyi sensu lato with a special focus on the dynamics of the plasmidome. Twenty-four genomes were sequenced from strains selected to represent as much as possible the genetic diversity in C. novyi sensu lato. Sixty-one plasmids were identified in these genomes and 28 of them were completed. The genomic comparisons revealed four separate lineages, which did not strictly correlate with the species designations. The plasmids were categorized into 13 different plasmid groups on the basis of their similarity and conservation of plasmid replication or partitioning genes. The plasmid groups, lineages and species were to a large extent entwined because plasmids and toxin genes had moved across the lineage boundaries. This dynamic process appears to be primarily driven by phages. We here present a comprehensive characterization of the complex species group C. novyi sensu lato, explaining the intermixed genetic properties. This study also provides examples how the reorganization of the botulinum toxin and the novyi alpha toxin genes within the plasmidome has affected the pathogenesis of the strains.

  10. Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens.

    Directory of Open Access Journals (Sweden)

    Hanna Skarin

    Full Text Available Clostridium botulinum (group III, Clostridium novyi and Clostridium haemolyticum are well-known pathogens causing animal botulism, gas gangrene/black disease, and bacillary hemoglobinuria, respectively. A close genetic relationship exists between the species, which has resulted in the collective term C. novyi sensu lato. The pathogenic traits in these species, e.g., the botulinum neurotoxin and the novyi alpha toxin, are mainly linked to a large plasmidome consisting of plasmids and circular prophages. The plasmidome of C. novyi sensu lato has so far been poorly characterized. In this study we explored the genomic relationship of a wide range of strains of C. novyi sensu lato with a special focus on the dynamics of the plasmidome. Twenty-four genomes were sequenced from strains selected to represent as much as possible the genetic diversity in C. novyi sensu lato. Sixty-one plasmids were identified in these genomes and 28 of them were completed. The genomic comparisons revealed four separate lineages, which did not strictly correlate with the species designations. The plasmids were categorized into 13 different plasmid groups on the basis of their similarity and conservation of plasmid replication or partitioning genes. The plasmid groups, lineages and species were to a large extent entwined because plasmids and toxin genes had moved across the lineage boundaries. This dynamic process appears to be primarily driven by phages. We here present a comprehensive characterization of the complex species group C. novyi sensu lato, explaining the intermixed genetic properties. This study also provides examples how the reorganization of the botulinum toxin and the novyi alpha toxin genes within the plasmidome has affected the pathogenesis of the strains.

  11. Common Mesophilic Anaerobes, Including Clostridium botulinum and Clostridium tetani, in 21 Soil Specimens

    Science.gov (United States)

    Smith, Louis Ds.

    1975-01-01

    A relatively rich medium was markedly superior to a dilute medium for the isolation of anaerobic bacteria from soil. The obligate anaerobes isolated from 21 soil samples were all clostridia and the counts ranged from 2.7 × 102 to 3.3 × 106 per g. The organisms most frequently isolated were Clostridium subterminale, C. sordellii, C. sporogenes, C. indolis, C. bifermentans, C. mangenoti, and C. perfringens. Seventeen other species were also recognized but almost one-third of the isolates could not be identified with any known species of Clostridum. C. botulinum type A was demonstrated in six soil samples, and type B in one. These soils were neutral to alkaline in reaction (average pH 7.9) and low in organic matter content (1.4%). The association of C. botulinum types A and B with neutral to alkaline soils was statistically significant (P = 0.001) as was their association with soils low in organic matter (P = 0.005). C. botulinum types E and F were found in one soil sample, pH 4.5, with organic matter 13.7%. C. tetani was isolated from two soil samples, both of intermediate pH value and higher than average organic matter content. PMID:238468

  12. Functional characterisation of germinant receptors in Clostridium botulinum and Clostridium sporogenes presents novel insights into spore germination systems.

    Science.gov (United States)

    Brunt, Jason; Plowman, June; Gaskin, Duncan J H; Itchner, Manoa; Carter, Andrew T; Peck, Michael W

    2014-09-01

    Clostridium botulinum is a dangerous pathogen that forms the highly potent botulinum toxin, which when ingested causes a deadly neuroparalytic disease. The closely related Clostridium sporogenes is occasionally pathogenic, frequently associated with food spoilage and regarded as the non-toxigenic equivalent of Group I C. botulinum. Both species form highly resistant spores that are ubiquitous in the environment and which, under favourable growth conditions germinate to produce vegetative cells. To improve the control of botulinum neurotoxin-forming clostridia, it is imperative to comprehend the mechanisms by which spores germinate. Germination is initiated following the recognition of small molecules (germinants) by a specific germinant receptor (GR) located in the spore inner membrane. The present study precisely defines clostridial GRs, germinants and co-germinants. Group I C. botulinum ATCC3502 contains two tricistronic and one pentacistronic GR operons, while C. sporogenes ATCC15579 has three tricistronic and one tetracistronic GR operons. Insertional knockout mutants, allied with characterisation of recombinant GRs shows for the first time that amino acid stimulated germination in C. botulinum requires two tri-cistronic encoded GRs which act in synergy and cannot function individually. Spore germination in C. sporogenes requires one tri-cistronic GR. Two other GRs form part of a complex involved in controlling the rate of amino-acid stimulated germination. The suitability of using C. sporogenes as a substitute for C. botulinum in germination studies and food challenge tests is discussed.

  13. Differential effects of sporulation temperature on the high pressure resistance of Clostridium botulinum type E spores and the interconnection with sporulation medium cation contents.

    Science.gov (United States)

    Lenz, Christian A; Vogel, Rudi F

    2015-04-01

    High pressure thermal (HPT) processing can be used to improve traditional preservation methods and increase food safety and durability, whereas quality related characteristics can be largely maintained. Clostridium (C.) botulinum type E is a non-proteolytic, psychrotrophic, toxin-producing spore former, commonly associated with aquatic environments in temperate regions of the northern hemisphere. Sporulation in nature is likely to occur under varying conditions including temperature and nutrient availability, which might affect resistance properties of resulting spores. In our study, we determined the effect of sporulation temperature (13-38 °C) on the resistance of three Clostridium botulinum type E strains to differently intense HPT treatments (200 MPa at 40 and 80 °C, and 800 MPa at 40 and 80 °C). Furthermore, the effect of cations on sporulation temperature-mediated alterations in HHP resistance was investigated. Results indicate that low and high sporulation temperatures can increase and decrease sporal HPT resistance, respectively, in a treatment-dependent (pressure level, treatment temperature) manner, whereas the trends observed are largely unaffected by pressure dwells (1 s-10 min). Furthermore, results show that the cation content of the sporulation medium (Ca(2+), Mg(2+), Mn(2+)) marginally influences and partially counteracts effects on the HPT resistance of spores grown at low and elevated temperatures, respectively. This suggests that sporulation temperature and medium cations provoke changes in some common spore resistance structures. Sporulation conditions can markedly affect spore resistance properties and, thus, should be considered for the experimental setup of worst case studies aiming to evaluate the effectiveness of food processes in terms of the inactivation of C. botulinum type E spores. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin.

    Science.gov (United States)

    Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Azarnia Tehran, Domenico; Montecucco, Cesare; Barth, Holger

    2016-04-01

    The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins.

  15. Efficacy of Clostridium botulinum types C and D toxoid vaccination in Danish cows.

    Science.gov (United States)

    Krüger, Monika; Skau, Marie; Shehata, Awad Ali; Schrödl, Wieland

    2013-10-01

    In the present study the efficacy Botulism vaccine (formalinised aluminum hydroxide gel adsorbed toxoid of Clostridium botulinum types C and D) was evaluated in four Danish dairy cows under field conditions. Other four dairy herds were unvaccinated. Blood serum of all animals was analyzed for specific C. botulinum types A, B, C, D and E antibodies using a developed ELISA. Feces of all animals were analyzed for botulinum neurotoxins (BoNTs) and C. botulinum spores. C. botulinum types C and D antibodies were significantly (p vaccinated animals. Vaccination with botulism vaccine significantly reduced (p vaccination increases specific blood serum antibodies and reduces free BoNTs and C. botulinum spores in feces. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Clostridium botulinum group I strain genotyping by 15-locus multilocus variable-number tandem-repeat analysis

    NARCIS (Netherlands)

    Fillo, S.; Giordani, F.; Anniballi, F.; Gorgé, O.; Ramisse, V.; Vergnaud, G.; Riehm, J.M.; Scholz, H.C.; Splettstoesser, W.D.; Kieboom, J.; Olsen, J.-S.; Fenicia, L.; Lista, F.

    2011-01-01

    Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of

  17. Detection of toxigenic Clostridium perfringens and Clostridium botulinum from food sold in Lagos, Nigeria.

    Science.gov (United States)

    Chukwu, Emelda E; Nwaokorie, Francisca O; Coker, Akitoye O; Avila-Campos, Mario J; Solis, Rosa L; Llanco, Luis A; Ogunsola, Folasade T

    2016-12-01

    Food-borne diseases contribute to the huge burden of sickness and death globally and in the last decade, have become more frequently reported in Africa. In line with this, food safety is becoming a significant and growing public health problem in Nigeria. Diarrhoea is a common problem in Nigeria and has been reported but there has been little data on the possibility of clostridia as aetiological agents. Clostridium species are ubiquitous in the environment and in the gastrointestinal tract of man and animals and can serve as a marker for faecal contamination. We set out to determine the potential of these foods to transmit Clostridium species. A total of 220 food commodities from six local governments in Lagos State were sampled. Isolates obtained were identified based on cultural, morphological and biochemical characteristics. Toxinotyping was done using multiplex-PCR with primers specific for alpha, beta, epsilon and iota-toxin genes, enterotoxigenic cpe gene and neurotoxigenic BoNt gene. Fifty (22.7%) clostridial species were isolated of which 29 (58%) were identified as C. perfringens. Toxinotyping of the 29 strains showed that 28 (96.6%) were toxin producing C. perfringens type A while one (3.4%) was C. perfringens type D. Two (4%) C. botulinum species were isolated and identified by 16S rRNA sequencing, both harbouring BoNt/A gene. The contamination rates of food with Clostridium species show that food hygiene is a problem and Clostridium species may be a source of food borne disease in Lagos State, Nigeria. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. [Clinical analysis of 53 patients with Clostridium botulinum food poisoning].

    Science.gov (United States)

    Zhang, Peng; Dong, Jianguang; Bai, Lili; Qiu, Zewu

    2017-05-01

    To analyze the epidemiologic data of patients with Clostridium botulinum food poisoning, and to improve the understanding, diagnosis and treatment of food borne botulism. A retrospective study was conducted. Fifty-three patients with Clostridium botulinum food poisoning admitted to Chinese PLA Center of Poisoning and Treatment from January 2009 to December 2016 were enrolled, and they were divided into mild, moderate, and severe groups according to the severity of disease. The clinical data including medical history, epidemiology data, routine blood test and blood biochemistry at hospital admission, the vital signs and arterial blood gas analysis before and after treatment, as well as the occurrence frequency of symptom and sign on set were collected. Fifty-three patients with food borne botulism were enrolled, with 33 patients in mild group, 13 in moderate group, and 7 in severe group. Most of the patients were female, the age distribution was in large span, the outbreak of disease was in groups mainly with the family, and patients were mainly located in Hebei Province, Beijing and Henan Province. The outbreaks were mainly happened in Spring and Summer, and homemade fermentation products were still the first cause of poisoning with the average latent period of (51.01±4.78) hours. The majority of patients with botulism were in mild resulted from the type A toxin. With the aggravation of disease, hospitalization time was gradually increased, white blood cell (WBC) and neutrophils (NEUT) at hospitalization admission, and respiratory rate (RR), heart rate (HR), fraction of inspired oxygen (FiO 2 ) before the treatment were shown in obviously rising trend, albumin (ALB) at hospitalization admission and pH, arterial partial pressure of oxygen (PaO2), arterial oxygen saturation (SaO 2 ) before treatment were in decline. The parameters in severe group were most severe, and had significant differences as compared with those of mild group [hospitalization time (days): 72

  19. Invasive slug populations (Arion vulgaris) as potential vectors for Clostridium botulinum.

    Science.gov (United States)

    Gismervik, Kristine; Bruheim, Torkjel; Rørvik, Liv M; Haukeland, Solveig; Skaar, Ida

    2014-10-03

    Norwegian meadows, including those for silage production, are recently found heavily invaded by the slug Arion vulgaris in exposed areas. As a consequence, large numbers of slugs might contaminate grass silage and cause a possible threat to animal feed quality and safety. It is well known that silage contaminated by mammalian or avian carcasses can lead to severe outbreaks of botulism among livestock. Invertebrates, especially fly-larvae (Diptera), are considered important in the transfer of Clostridium botulinum type C and its toxins among birds in wetlands. C. botulinum form highly resistant spores that could easily be consumed by the slugs during feeding. This study aimed to determine whether Arion vulgaris could hold viable C. botulinum and enrich them, which is essential knowledge for assessing the risk of botulism from slug-contaminated silage. Slug carcasses, slug feces and live slugs were tested by a quantitative real-time PCR (qPCR) method after being fed ≅ 5.8 × 104 CFU C. botulinum type C spores/slug. Low amounts of C. botulinum were detected by qPCR in six of 21 slug carcasses with an even spread throughout the 17 day long experiment. Declining amounts of C. botulinum were excreted in slug feces up to day four after the inoculated feed was given. C. botulinum was only quantified the first two days in the sampling of live slugs. The viability of C. botulinum was confirmed for all three sample types (slug carcasses, slug feces and live slugs) by visible growth in enrichment media combined with obtaining a higher quantification cycle (Cq) value than from the non-enriched samples. Neither dead nor live invasive Arion vulgaris slugs were shown to enrich Clostridium botulinum containing the neurotoxin type C gene in this study. Slugs excreted viable C. botulinum in their feces up to day four, but in rapidly decreasing numbers. Arion vulgaris appear not to support enrichment of C. botulinum type C.

  20. Clostridium botulinum group III: a group with dual identity shaped by plasmids, phages and mobile elements

    OpenAIRE

    Skarin, Hanna; Håfström, Therese; Westerberg, Josefina; Segerman, Bo

    2011-01-01

    Abstract Background Clostridium botulinum strains can be divided into four physiological groups that are sufficiently diverged to be considered as separate species. Here we present the first complete genome of a C. botulinum strain from physiological group III, causing animal botulism. We also compare the sequence to three new draft genomes from the same physiological group. Results The 2.77 Mb chromosome was highly conserved between the isolates and also closely related to that of C. novyi. ...

  1. Effect of media, additives, and incubation conditions on the recovery of high pressure and heat-injured Clostridium botulinum spores.

    Science.gov (United States)

    Reddy, N R; Tetzloff, R C; Skinner, G E

    2010-08-01

    The effect of additives and post-treatment incubation conditions on the recovery of high pressure and heat-injured (i.e., processed at 620 MPa and 95 and 100 degrees C for 5 min) spores of Clostridium botulinum strains, 62-A (proteolytic type A) and 17-B (nonproteolytic type B) was studied. High pressure and heat-injured spores were inoculated into TPGY (Trypticase-Peptone-Glucose-Yeast extract) anaerobic broth media containing additives (lysozyme, L-alanine, L-aspartic acid, dipicolonic acid, sodium bicarbonate, and sodium lactate) at various concentrations (0-10 microg/ml) individually or in combination. The spore counts of high pressure and heat-injured 62-A and 17-B recovered from TPGY broth containing lysozyme (10 microg/ml) incubated for 4 months versus that recovered from peptone-yeast extract-glucose-starch (PYGS) plating agar containing lysozyme (10 microg/ml) incubated under anaerobic conditions for 5 days were also compared. None of the additives either individually or in combination in TPGY broth improved recovery of injured spore enumeration compared to processed controls without additives. Addition of lysozyme at concentrations of 5 and 10 microg/ml in TPGY broth improved initial recovery of injured spores of 17-B during the first 4 days of incubation but did not result in additional recovery at the end of the 4 month incubation compared to the processed control without lysozyme. Adding lysozyme at a concentration of 10 microg/ml to PYGS plating agar resulted in no effect on the recovery of high pressure and heat-injured 62-A and 17-B spores. The recovery counts of high pressure and heat-injured spores of 62-A and 17-B were lower (i.e., <1.0 log units) with PYGS plating agar compared to the MPN method using TPGY broth as the growth medium. Published by Elsevier Ltd.

  2. Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System.

    Science.gov (United States)

    Bradshaw, Marite; Tepp, William H; Whitemarsh, Regina C M; Pellett, Sabine; Johnson, Eric A

    2014-12-01

    Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing C. botulinum strain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in trans by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in Escherichia coli has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  3. Diversity of the Germination Apparatus in Clostridium botulinum Groups I, II, III and IV

    Directory of Open Access Journals (Sweden)

    Jason Brunt

    2016-10-01

    Full Text Available Clostridium botulinum is a highly dangerous pathogen that forms very resistant endospores that are ubiquitous in the environment, and which, under favourable conditions germinate to produce vegetative cells that multiply and form the exceptionally potent botulinum neurotoxin. To improve the control of botulinum neurotoxin-forming clostridia, it is important to understand the mechanisms involved in spore germination. Here we present models for spore germination in C. botulinum based on comparative genomics analyses, with C. botulinum Groups I and III sharing similar pathways, which differ from those proposed for C. botulinum Groups II and IV. All spores germinate in response to amino acids interacting with a germinant receptor, with four types of germinant receptor identified (encoded by various combinations of gerA, gerB and gerC genes (gerX. There are three gene clusters with an ABC-like configuration; ABC gerX1, ABABCB gerX2 and ACxBBB gerX4, and a single CA-B gerX3 gene cluster. Subtypes have been identified for most germinant receptors types, and the individual GerX subunits of each cluster show similar grouping in phylogenetic trees. C. botulinum Group I contained the largest variety of gerX subtypes, with three gerX1, three gerX2 and one gerX3 subtypes, while C. botulinum Group III contained two gerX1 types and one gerX4. C. botulinum Groups II and IV contained a single germinant receptor, gerX3 and gerX1, respectively. It is likely that all four C. botulinum Groups include a SpoVA channel involved in DPA release. The cortex lytic enzymes present in C. botulinum Groups I and III appear to be CwlJ and SleB, while in C. botulinum Groups II and IV, SleC appears to be important.

  4. Clostridium botulinum Spores Found in Honey from Small Apiaries in Poland

    Directory of Open Access Journals (Sweden)

    Wojtacka Joanna

    2016-12-01

    Full Text Available A total of 102 honey samples collected from small apiaries (≤ 20 hives in Poland were analysed for the presence of Clostridium botulinum spores. The samples were prepared using the dilution centrifugation method and cultured in parallel in cooked meat medium (CMM and tripticase peptone glucose yeast (TPGY enrichment broths. Identification of toxin types A, B, and E of Clostridium botulinum strains was performed with the use of the multiplex PCR method. Positive samples were also subjected to quantitative analysis with the use of Clostridium botulinum Isolation Agar Base (CBAB. The prevalence analysis showed 22 (21.6% samples contaminated with C. botulinum spores. The major serotype detected was botulin neurotoxin type A – 16 (72.7% whereas type B was found in 3 (13.6% honey samples and type E also only in 3 (13.6% honey samples. Dual-toxin-producing strains were noted. The average quantity of spores in PCR - C. botulinum positive samples was 190 in 1 gram of honey.

  5. Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification

    NARCIS (Netherlands)

    Barth, H; Blocker, D; Behlke, J; Bergsma-Schutter, W; Brisson, A; Benz, R; Aktories, K

    2000-01-01

    The actin-ADP-ribosylating binary Clostridium botulinum C2 toxin consists of two individual proteins, the binding/translocation component C2II and the enzyme component C2I. To elicit its cytotoxic action, C2II binds to a receptor on the cell surface and mediates cell entry of C2I via

  6. Neurotoxin gene profiling of Clostridium botulinum types C and D gathered from different countries within Europe

    NARCIS (Netherlands)

    Woudstra, C.; Skarin, A.; Anniballi, F.; Fenicia, F.; Bano, L.; Drigo, I.; Koene, M.G.J.; Bäyon-Auboyer, M.H.; Buffereau, J.P.; Medici, D.; Fach, P.

    2012-01-01

    Clostridium botulinum types C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous

  7. Behaviour of individual spores of non proteolytic Clostridium botulinum as an element in quantitative risk assessment

    NARCIS (Netherlands)

    Smelt, J.P.; Stringer, S.C.; Brul, S.

    2013-01-01

    Botulism is a true food poisoning hazard. It is caused by a deadly neurotoxin produced in food by the spore forming organism Clostridium botulinum, which is found in soil all over the world. The frequency of botulism cases is rare, but each year a number of them occur. Ideally a quantitative

  8. Clostridium botulinum type E occurs and grows in the alga Cladophora glomerata

    Science.gov (United States)

    Byappanahalli, M.N.; Whitman, R.L.

    2009-01-01

    In recent years, massive avian die-offs from Clostridium botulinum type E infection have occurred in the Sleeping Bear Dunes National Lakeshore (SLBE) area of Lake Michigan. These outbreaks have been coincidental with massive blooms of the green algae Cladophora, mostly Cladophora glomerata. We tested the hypothesis that Clostridium botulinum type E can grow under suitable conditions in these algal mats. In a lab mesocosm study, Cladophora from four outbreak-impacted beaches from SLBE were compared with four unimpacted beaches in the Milwaukee–Racine area for bontE gene of Clostridium botulinum. Frequency of the bontE gene was higher after incubation (25 °C for up to 6 weeks) of Cladophora from impacted vs. the unimpacted area. Since no type E gene was detected initially in Cladophora from any of the eight locations, we infer that the increased occurrence of type E gene arose from spore germination or vegetative Clostridium growth within the existing algal mats of SLBE. Moreover, we found that the congener Clostridium perfringens readily grows in mesocosms containing Cladophora.

  9. Diversity of Group I and II Clostridium botulinum Strains from France Including Recently Identified Subtypes.

    Science.gov (United States)

    Mazuet, Christelle; Legeay, Christine; Sautereau, Jean; Ma, Laurence; Bouchier, Christiane; Bouvet, Philippe; Popoff, Michel R

    2016-06-13

    In France, human botulism is mainly food-borne intoxication, whereas infant botulism is rare. A total of 99 group I and II Clostridium botulinum strains including 59 type A (12 historical isolates [1947-1961], 43 from France [1986-2013], 3 from other countries, and 1 collection strain), 31 type B (3 historical, 23 recent isolates, 4 from other countries, and 1 collection strain), and 9 type E (5 historical, 3 isolates, and 1 collection strain) were investigated by botulinum locus gene sequencing and multilocus sequence typing analysis. Historical C. botulinum A strains mainly belonged to subtype A1 and sequence type (ST) 1, whereas recent strains exhibited a wide genetic diversity: subtype A1 in orfX or ha locus, A1(B), A1(F), A2, A2b2, A5(B2') A5(B3'), as well as the recently identified A7 and A8 subtypes, and were distributed into 25 STs. Clostridium botulinum A1(B) was the most frequent subtype from food-borne botulism and food. Group I C. botulinum type B in France were mainly subtype B2 (14 out of 20 historical and recent strains) and were divided into 19 STs. Food-borne botulism resulting from ham consumption during the recent period was due to group II C. botulinum B4. Type E botulism is rare in France, 5 historical and 1 recent strains were subtype E3. A subtype E12 was recently identified from an unusual ham contamination. Clostridium botulinum strains from human botulism in France showed a wide genetic diversity and seems to result not from a single evolutionary lineage but from multiple and independent genetic rearrangements. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  10. Implications of Genome-Based Discrimination between Clostridium botulinum Group I and Clostridium sporogenes Strains for Bacterial Taxonomy.

    Science.gov (United States)

    Weigand, Michael R; Pena-Gonzalez, Angela; Shirey, Timothy B; Broeker, Robin G; Ishaq, Maliha K; Konstantinidis, Konstantinos T; Raphael, Brian H

    2015-08-15

    Taxonomic classification of Clostridium botulinum is based on the production of botulinum neurotoxin (BoNT), while closely related, nontoxic organisms are classified as Clostridium sporogenes. However, this taxonomic organization does not accurately mirror phylogenetic relationships between these species. A phylogenetic reconstruction using 2,016 orthologous genes shared among strains of C. botulinum group I and C. sporogenes clearly separated these two species into discrete clades which showed ∼93% average nucleotide identity (ANI) between them. Clustering of strains based on the presence of variable orthologs revealed 143 C. sporogenes clade-specific genetic signatures, a subset of which were further evaluated for their ability to correctly classify a panel of presumptive C. sporogenes strains by PCR. Genome sequencing of several C. sporogenes strains lacking these signatures confirmed that they clustered with C. botulinum strains in a core genome phylogenetic tree. Our analysis also identified C. botulinum strains that contained C. sporogenes clade-specific signatures and phylogenetically clustered with C. sporogenes strains. The genome sequences of two bont/B2-containing strains belonging to the C. sporogenes clade contained regions with similarity to a bont-bearing plasmid (pCLD), while two different strains belonging to the C. botulinum clade carried bont/B2 on the chromosome. These results indicate that bont/B2 was likely acquired by C. sporogenes strains through horizontal gene transfer. The genome-based classification of these species used to identify candidate genes for the development of rapid assays for molecular identification may be applicable to additional bacterial species that are challenging with respect to their classification. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Systemic colonization of clover (Trifolium repens by Clostridium botulinum strain 2301

    Directory of Open Access Journals (Sweden)

    Matthias eZeiller

    2015-10-01

    Full Text Available In recent years, cases of botulism in cattle and other farm animals and also in farmers increased dramatically. It was proposed, that these cases could be affiliated with the spreading of compost or other organic manures contaminated with Clostridium botulinum spores on farm land. Thus, soils and fodder plants and finally farm animals could be contaminated. Therefore, the colonization behavior and interaction of the botulinum neurotoxin (BoNT D producing C. botulinum strain 2301 and the non-toxin producing Clostridium sporogenes strain 1739 were investigated on clover (Trifolium repens in a field experiment as well as in phytochamber experiments applying axenic and additionally soil based systems under controlled conditions. Plants were harvested and divided into root and shoot parts for further DNA isolation and PCR assays; subsamples were fixed for fluorescence in situ hybridization (FISH analysis in combination with confocal laser scanning microscopy (CLSM. To target C. botulinum and C. sporogenes, 16S rDNA directed primers were used and to specifically detect C. botulinum, BoNT D toxin genes targeted primers, using a multiplex PCR approach, were applied. Our results demonstrate an effective colonization of roots and shoots of clover by C. botulinum strain 2301 and C. sporogenes strain 1739. Detailed analysis of colonization behavior showed that C. botulinum can occur as individual cells, in cell clusters and in microcolonies within the rhizosphere, lateral roots and within the roots tissue of clover. In addition, we observed significant differences in the growth behavior of clover plants when inoculated with Clostridia spores, indicating a plant growth promoting effect. Inoculated plants showed an increased growth index (shoot size, wet and dry weight and an enlarged root system, which suggests the involvement of phytohormonal effects induced by the systemic colonization of clover by C. botulinum strain 2301.

  12. Systemic colonization of clover (Trifolium repens) by Clostridium botulinum strain 2301.

    Science.gov (United States)

    Zeiller, Matthias; Rothballer, Michael; Iwobi, Azuka N; Böhnel, Helge; Gessler, Frank; Hartmann, Anton; Schmid, Michael

    2015-01-01

    In recent years, cases of botulism in cattle and other farm animals and also in farmers increased dramatically. It was proposed, that these cases could be affiliated with the spreading of compost or other organic manures contaminated with Clostridium botulinum spores on farm land. Thus, soils and fodder plants and finally farm animals could be contaminated. Therefore, the colonization behavior and interaction of the botulinum neurotoxin (BoNT D) producing C. botulinum strain 2301 and the non-toxin producing Clostridium sporogenes strain 1739 were investigated on clover (Trifolium repens) in a field experiment as well as in phytochamber experiments applying axenic and additionally soil based systems under controlled conditions. Plants were harvested and divided into root and shoot parts for further DNA isolation and polymerase chain reaction (PCR) assays; subsamples were fixed for fluorescence in situ hybridization analysis in combination with confocal laser scanning microscopy. In addition, we observed significant differences in the growth behavior of clover plants when inoculated with clostridial spores, indicating a plant growth promoting effect. Inoculated plants showed an increased growth index (shoot size, wet and dry weight) and an enlarged root system induced by the systemic colonization of clover by C. botulinum strain 2301. To target C. botulinum and C. sporogenes, 16S rDNA directed primers were used and to specifically detect C. botulinum, BoNT D toxin genes targeted primers, using a multiplex PCR approach, were applied. Our results demonstrate an effective colonization of roots and shoots of clover by C. botulinum strain 2301 and C. sporogenes strain 1739. Detailed analysis of colonization behavior showed that C. botulinum can occur as individual cells, in cell clusters and in microcolonies within the rhizosphere, lateral roots and within the roots tissue of clover.

  13. In situ detection of the Clostridium botulinum type C1 toxin gene in wetland sediments with a nested PCR assay

    Science.gov (United States)

    Williamson, Judy L.; Rocke, Tonie E.; Aiken, Judd M.

    1999-01-01

    A nested PCR was developed for detection of the Clostridium botulinum type C1 toxin gene in sediments collected from wetlands where avian botulism outbreaks had or had not occurred. The C1 toxin gene was detected in 16 of 18 sites, demonstrating both the ubiquitous distribution of C. botulinum type C in wetland sediments and the sensitivity of the detection assay.

  14. Molucular gene profiling of Clostridium botulinum group III and their detection in naturally contaminated samples originating from various European countries

    NARCIS (Netherlands)

    Woudstra, C.; LeMarechal, C.; Souillard, R.; Bäyon-Auboyer, M.H.; Anniballi, F.; Auricchio, B.; Medici, De D.; Bano, L.; Koene, M.G.J.; Sansonetti, M.H.; Hansbauer, E.M.; Desoutter, D.; Dorner, M.B.; Fach, P.; Dorner, B.G.

    2015-01-01

    We report the development of real-time PCR assays for genotyping Clostridium botulinum group III targeting the newly defined C. novyi sensu lato group; the nontoxic nonhemagglutinin (NTNH)-encoding gene ntnh; the botulinum neurotoxin (BoNT)-encoding genes bont/C, bont/C/D, bont/D, and bont/D/C; and

  15. Hemagglutinin gene shuffling among Clostridium botulinum serotypes C and D yields distinct sugar recognition of the botulinum toxin complex.

    Science.gov (United States)

    Miyata, Keita; Suzuki, Tomonori; Hayashi, Shintaro; Miyashita, Shin-Ichiro; Ohyama, Tohru; Niwa, Koichi; Watanabe, Toshihiro; Sagane, Yoshimasa

    2015-10-01

    Clostridium botulinum strains produce a large-sized toxin complex (TC) that is composed of botulinum neurotoxin (BoNT), non-toxic non-hemagglutinin and three different hemagglutinins (HA-70, HA-33 and HA-17). HA components enhance toxin delivery across the intestinal cell wall in a sugar chain-dependent manner. Here we characterized the sugar recognition of serotype D strain 1873 (D-1873) botulinum L-TC. Most L-TCs produced by serotype C and D strains bind to cells via interactions between HA-33 and cell surface sialo-oligosaccharides. However, like the previously reported L-TC produced by serotype C strain Yoichi (C-Yoichi), D-1873 L-TC binds only to cells that have been treated with neuraminidase, indicating that they recognize asialo-oligosaccharides. The D-1873 HA-33 amino acid sequence is similar to that of C-Yoichi, but had lower similarity to the majority of serotype C and D HA-33s. A comparison of TC component primary structures for 12 serotype C and D strains suggested that at least three types of HA-33 genes exist, and these are shuffled among the serotype C and D strains independently of BoNT serotype. This shuffling produces the distinct sugar recognition of serotype C and D botulinum TCs. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Glyphosate suppresses the antagonistic effect of Enterococcus spp. on Clostridium botulinum.

    Science.gov (United States)

    Krüger, Monika; Shehata, Awad Ali; Schrödl, Wieland; Rodloff, Arne

    2013-04-01

    During the last 10-15 years, an increase of Clostridium botulinum associated diseases in cattle has been observed in Germany. The reason for this development is currently unknown. The normal intestinal microflora is a critical factor in preventing intestinal colonisation by C. botulinum as shown in the mouse model of infant botulism. Numerous bacteria in the gastro-intestinal tract (GIT) produce bacteriocines directed against C. botulinum and other pathogens: Lactic acid producing bacteria (LAB) such as lactobacilli, lactococci and enterococci, generate bacteriocines that are effective against Clostridium spp. A reduction of LAB in the GIT microbiota by ingestion of strong biocides like glyphosate could be an explanation for the observed increase in levels of C. botulinum associated diseases. In the present paper, we report on the toxicity of glyphosate to the most prevalent Enterococcus spp. in the GIT. Ingestion of this herbicide could be a significant predisposing factor that is associated with the increase in C. botulinum mediated diseases in cattle. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. The inhibition of Clostridium botulinum type C by other bacteria in wetland sediments

    Science.gov (United States)

    Sandler, Renee J.; Rocke, Tonie E.; Yuill, Thomas M.

    1998-01-01

    Bacteria with inhibitory activity against Clostridium botulinum type C were isolated from 32% of sediment samples (n = 1600) collected from 10 marshes in a northern California wetland over a 12 mo period. Aerobic and anaerobic bacteria with inhibitory activity were isolated from 12% and 23% of the samples, respectively. Bacteria with inhibitory activity were isolated from all 10 study sites and throughout the year. This study demonstrates that bacteria with inhibitory activity against C. botulinum type C occur naturally in wetland sediments.

  18. Identification and genetic characterization of Clostridium botulinum serotype A strains from commercially pasteurized carrot juice.

    Science.gov (United States)

    Marshall, Kristin M; Nowaczyk, Louis; Raphael, Brian H; Skinner, Guy E; Rukma Reddy, N

    2014-12-01

    Clostridium botulinum is an important foodborne pathogen capable of forming heat resistant endospores and producing deadly botulinum neurotoxins (BoNTs). In 2006, C. botulinum was responsible for an international outbreak of botulism attributed to the consumption of commercially pasteurized carrot juice. The purpose of this study was to isolate and characterize strains of C. botulinum from the adulterated product. Carrot juice bottles retrieved from the manufacturing facility were analyzed for the presence of BoNT and BoNT-producing isolates using DIG-ELISA. Toxigenic isolates from the carrot juice were analyzed using pulsed-field gel electrophoresis (PFGE) and DNA microarray analysis to determine their genetic relatedness to the original outbreak strains CDC51348 and CDC51303. PFGE revealed that isolates CJ4-1 and CJ10-1 shared an identical pulsotype with strain CDC51303, whereas isolate CJ5-1 displayed a unique restriction banding pattern. DNA microarray analysis identified several phage related genes unique to strain CJ5-1, and Southern hybridization analysis of XhoI digested and nondigested DNA showed their chromosomal location, while a homolog to pCLI_A009 of plasmid pCLI of C. botulinum serotype Langeland F, was located on a small plasmid. The acquisition or loss of bacteriophages and other mobile genetic elements among C. botulinum strains has epidemiological and evolutionary implications. Published by Elsevier Ltd.

  19. Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum.

    Science.gov (United States)

    Anniballi, Fabrizio; Auricchio, Bruna; Delibato, Elisabetta; Antonacci, Monia; De Medici, Dario; Fenicia, Lucia

    2012-01-27

    Clostridium botulinum type C and type D belonging to the group III organisms, are mainly responsible for animal botulism outbreaks. Clinical signs alone are often insufficient to make a diagnosis of botulism and a laboratory confirmation is required. Laboratory confirmation can be performed by demonstrating the presence of botulinum neurotoxins in serum, gastrointestinal contents, liver, wound of sick or dead animals, or by demonstrating the presence of C. botulinum in gastrointestinal contents, liver, and wound. Demonstration of spores in gastrointestinal contents or tissue of animals with clinical signs indicative of botulism reinforces the clinical diagnosis. With the aim of detecting and typing C. botulinum group III organisms, a multiplex real-time PCR SYBR Green was developed and in-house validated. Selectivity, limit of detection, relative accuracy, relative specificity, relative sensitivity, and repeatability of the method were investigated. The multiplex real-time PCR SYBR green used showed a 100% selectivity, 100% relative accuracy, 100% relative specificity, 100% relative sensitivity and a limit of detection of 277 and 580 DNA copies for C. botulinum type C and C. botulinum type D, respectively. The method reported here represents a suitable tool for laboratory diagnosis of type C and D botulism and for testing a large number of samples collected during the animal botulism surveillance and prevention activities. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Evidence that plasmid-borne botulinum neurotoxin type B genes are widespread among Clostridium botulinum serotype B strains.

    Directory of Open Access Journals (Sweden)

    Giovanna Franciosa

    Full Text Available BACKGROUND: Plasmids that encode certain subtypes of the botulinum neurotoxin type B have recently been detected in some Clostridium botulinum strains. The objective of the present study was to investigate the frequency with which plasmid carriage of the botulinum neurotoxin type B gene (bont/B occurs in strains of C. botulinum type B, Ab, and A(B, and whether plasmid carriage is bont/B subtype-related. METHODOLOGY/PRINCIPAL FINDINGS: PCR-Restriction fragment length polymorphism was employed to identify subtypes of the bont/B gene. Pulsed-field gel electrophoresis and Southern blot hybridization with specific probes were performed to analyze the genomic location of the bont/B subtype genes. All five known bont/B subtype genes were detected among the strains; the most frequently detected subtype genes were bont/B1 and /B2. Surprisingly, the bont/B subtype gene was shown to be plasmid-borne in >50% of the total strains. The same bont/B subtype gene was associated with the chromosome in some strains, whereas it was associated with a plasmid in others. All five known bont/B subtype genes were in some cases found to reside on plasmids, though with varying frequency (e.g., most of the bont/B1 subtype genes were located on plasmids, whereas all but one of the bont/B2 subtypes were chromosomally-located. Three bivalent isolates carried both bont/A and /B genes on the same plasmid. The plasmids carrying the bont gene were five different sizes, ranging from approximately 55 kb to approximately 245 kb. CONCLUSIONS/SIGNIFICANCE: The unexpected finding of the widespread distribution of plasmids harboring the bont/B gene among C. botulinum serotype B strains provides a chance to examine their contribution to the dissemination of the bont genes among heterogeneous clostridia, with potential implications on issues related to pathogenesis and food safety.

  1. Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains.

    Science.gov (United States)

    Janganan, Thamarai K; Mullin, Nic; Tzokov, Svetomir B; Stringer, Sandra; Fagan, Robert P; Hobbs, Jamie K; Moir, Anne; Bullough, Per A

    2016-10-01

    Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75-100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Conditions associated with Clostridium sporogenes growth as a surrogate for Clostridium botulinum in nonthermally processed canned butter.

    Science.gov (United States)

    Taylor, R H; Dunn, M L; Ogden, L V; Jefferies, L K; Eggett, D L; Steele, F M

    2013-05-01

    The objective of this study was to better understand the effect of butter composition and emulsion structure on growth and survival of Clostridium sporogenes, used as a surrogate for C. botulinum in canned butter. The lack of a thermal process step in commercially available canned butter raises questions of potential safety, because it is hermetically sealed and generally exhibits anaerobic growth conditions, which are optimal for Clostridium botulinum growth. Without thermal processing, low-acid canned foods must have inhibitory factors present to prevent C. botulinum growth. Some potential intrinsic inhibitory factors, or hurdles, within butter include: reduced water activity, acidity in cultured products, elevated salt content, and the micro-droplet nature of the aqueous phase in the butter emulsion. It was hypothesized that a normal, intact butter emulsion would have sufficient hurdles to prevent C. botulinum growth, whereas a broken butter emulsion would result in a coalesced aqueous phase that would allow for C. botulinum growth. Batch-churned butter was inoculated with C. sporogenes; butter samples with varying salt contents (0, 0.8, 1.6, and 2.4% wt/wt NaCl) were prepared and stored in coated steel cans for varying times (1 or 2 wk) and temperatures (22 or 41°C) to determine temperature and emulsion structure effects on C. sporogenes growth. Samples stored at 41°C showed a significant increase in C. sporogenes growth compared with those stored at 22°C. Furthermore, NaCl addition was found to have a significant effect on C. sporogenes growth, with 0.8% NaCl promoting more growth than 0%, but with decreases in growth observed at 1.6 and 2.4%. Uninoculated control plates were also found to have bacterial growth; this growth was attributed to other anaerobic bacteria present within the cream. It was concluded that removal of the hurdle created by the micro-droplet size of the emulsion aqueous phase could result in C. botulinum growth even at elevated salt

  3. Mechanisms of food processing and storage-related stress tolerance in Clostridium botulinum.

    Science.gov (United States)

    Dahlsten, Elias; Lindström, Miia; Korkeala, Hannu

    2015-05-01

    Vegetative cultures of Clostridium botulinum produce the extremely potent botulinum neurotoxin, and may jeopardize the safety of foods unless sufficient measures to prevent growth are applied. Minimal food processing relies on combinations of mild treatments, primarily to avoid deterioration of the sensory qualities of the food. Tolerance of C. botulinum to minimal food processing is well characterized. However, data on effects of successive treatments on robustness towards further processing is lacking. Developments in genetic manipulation tools and the availability of annotated genomes have allowed identification of genetic mechanisms involved in stress tolerance of C. botulinum. Most studies focused on low temperature, and the importance of various regulatory mechanisms in cold tolerance of C. botulinum has been demonstrated. Furthermore, novel roles in cold tolerance were shown for metabolic pathways under the control of these regulators. A role for secondary oxidative stress in tolerance to extreme temperatures has been proposed. Additionally, genetic mechanisms related to tolerance to heat, low pH, and high salinity have been characterized. Data on genetic stress-related mechanisms of psychrotrophic Group II C. botulinum strains are scarce; these mechanisms are of interest for food safety research and should thus be investigated. This minireview encompasses the importance of C. botulinum as a food safety hazard and its central physiological characteristics related to food-processing and storage-related stress. Special attention is given to recent findings considering genetic mechanisms C. botulinum utilizes in detecting and countering these adverse conditions. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  4. Evaluation of DNA Extraction Methods Suitable for PCR-based Detection and Genotyping of Clostridium botulinum

    DEFF Research Database (Denmark)

    Auricchio, Bruna; Anniballi, Fabrizio; Fiore, Alfonsina

    2013-01-01

    Sufficient quality and quantity of extracted DNA is critical to detecting and performing genotyping of Clostridium botulinum by means of PCR-based methods. An ideal extraction method has to optimize DNA yield, minimize DNA degradation, allow multiple samples to be extracted, and be efficient...... in terms of cost, time, labor, and supplies. Eleven botulinum toxin–producing clostridia strains and 25 samples (10 food, 13 clinical, and 2 environmental samples) naturally contaminated with botulinum toxin–producing clostridia were used to compare 4 DNA extraction procedures: Chelex® 100 matrix, Phenol......-Cloroform-Isoamyl alcohol, NucliSENS® magnetic extraction kit, and DNeasy® Blood & Tissue kit. Integrity, purity, and amount of amplifiable DNA were evaluated. The results show that the DNeasy® Blood & Tissue kit is the best extraction method evaluated because it provided the most pure, intact, and amplifiable DNA. However...

  5. The same clade of Clostridium botulinum strains is causing avian botulism in southern and northern Europe.

    Science.gov (United States)

    Anza, Ibone; Skarin, Hanna; Vidal, Dolors; Lindberg, Anna; Båverud, Viveca; Mateo, Rafael

    2014-04-01

    Avian botulism is a paralytic disease caused by Clostridium botulinum-produced botulinum neurotoxins (BoNTs), most commonly of type C/D. It is a serious disease of waterbirds and poultry flocks in many countries in Europe. The objective of this study was to compare the genetic relatedness of avian C. botulinum strains isolated in Spain with strains isolated in Sweden using pulsed-field gel electrophoresis (PFGE). Fifteen strains were isolated from Spanish waterbirds using an immunomagnetic separation technique. Isolates were characterized by PCR, and all were identified as the genospecies Clostridium novyi sensu lato and eight harboured the gene coding for the BoNT type C/D. PFGE analysis of the strains revealed four highly similar pulsotypes, out of which two contained strains from both countries. It also showed that outbreaks in wild and domestic birds can be caused by the same strains. These results support a clonal spreading of the mosaic C. botulinum type C/D through Europe and give relevant information for future epidemiological studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Pan-genome analysis of Clostridium botulinum reveals unique targets for drug development.

    Science.gov (United States)

    Bhardwaj, Tulika; Somvanshi, Pallavi

    2017-08-05

    Clostridium botulinum, a formidable pathogen is responsible for the emerging cause of food poisoning cases on the global canvas. The endemicity of bacterium Clostridium botulinum is reflected by the sudden hospital outbreaks and increased resistance towards multiple drugs. Therefore, a combined approach of in-silico comparative genomic analysis with statistical analysis was applied to overcome the limitation of bench-top technologies. Owing to the paucity of genomic data available by the advent of third generation sequencing technologies, several 'omics' technologies were applied to understand the underlying evolutionary pattern and lifestyle of the bacterial pathogen using phylogenomics. The calculation of pan-genome, core genome and singletons provides view of genetic repertoire of the bacterial pathogen lineage at the successive level, orthology shared and specific gene subsets. In addition, assessment of pathogenomic potential, resistome, toxin/antitoxin family in successive pathogenic strains of Clostridium botulinum aids in revealing more specific targets for drug design and development. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. DEVELOPMENT OF ENZYME-LINKAGE IMMUNOSORBENT ASSAY AGAINST TYPE B OF CLOSTRIDIUM BOTULINUM: A PRELIMINARY STUDY

    Directory of Open Access Journals (Sweden)

    S. N. Depamede

    2011-12-01

    Full Text Available Clostridium botulinum neurotoxin (BoNTs is one of the causes of economic loss in the livestock industry. This economic loss would be as a direct result when animals poisoned by BoNTs or indirectly when the livestock products are contaminated by BoNTs, which end up with the products are banned by authority. Therefore a routine surveillance of BoNTs in the farm and in livestock product processing industry is urgently needed. One of the most relatively quick and accurate methods to perform a routine detection of the presence of BoNTs is enzyme-linkage immunosorbant assay (ELISA. In this article we describe the results of the development of ELISA, using polyclonal antibodies against BoNTs-B produced locally. Antibodies were generated from six Balb/c mice with standard immunological methods. Mice were immunized three times for a period of 8 weeks with a commercial type B Clostridium botulinum toxoid at a dose of 100 ng per mouse per injection. The resulting antibody was purified by a combination of ammonium sulfate precipitation 50% (w/v technique and a protein A column method. The results of this preliminary study indicated that the developed ELISA method capable of detecting type B Clostridium botulinum toxin up to 1.0 ng/ml.

  8. Two-component systems are involved in the regulation of botulinum neurotoxin synthesis in Clostridium botulinum type A strain Hall.

    Directory of Open Access Journals (Sweden)

    Chloé Connan

    Full Text Available Clostridium botulinum synthesizes a potent neurotoxin (BoNT which associates with non-toxic proteins (ANTPs to form complexes of various sizes. The bont and antp genes are clustered in two operons. In C. botulinum type A, bont/A and antp genes are expressed during the end of the exponential growth phase and the beginning of the stationary phase under the control of an alternative sigma factor encoded by botR/A, which is located between the two operons. In the genome of C. botulinum type A strain Hall, 30 gene pairs predicted to encode two-component systems (TCSs and 9 orphan regulatory genes have been identified. Therefore, 34 Hall isogenic antisense strains on predicted regulatory genes (29 TCSs and 5 orphan regulatory genes have been obtained by a mRNA antisense procedure. Two TCS isogenic antisense strains showed more rapid growth kinetics and reduced BoNT/A production than the control strain, as well as increased bacterial lysis and impairment of the bacterial cell wall structure. Three other TCS isogenic antisense strains induced a low level of BoNT/A and ANTP production. Interestingly, reduced expression of bont/A and antp genes was shown to be independent of botR/A. These results indicate that BoNT/A synthesis is under the control of a complex network of regulation including directly at least three TCSs.

  9. Seasonal prevalence of Clostridium botulinum type C in the sediments of the northern California wetland

    Science.gov (United States)

    Sandler, Renee J.; Rocke, T.E.; Samuel, M.D.; Yuill, Thomas M.

    1993-01-01

    The prevalence of Clostridium botulinum type C (% of positive sediment samples) was determined in 10 marshes at Sacramento National Wildlife Refuge (SNWR), located in the Central Valley of California (USA), where avian botulism epizootics occur regularly. Fifty-two percent of 2,200 sediment samples collected over an 18-mo period contained C. botulinum type C (both neurotoxic and aneurotoxic) which was present throughout the year in all 10 marshes. The prevalence of C. botulinum type C was similar in marshes with either high or low botulism losses in the previous 5 yr. Marshes with avian botulism mortality during the study had similar prevalences as marshes with no mortality. However, the prevalence of C. botulinum type C was higher in marshes that remained flooded all year (permanent) compared with marshes that were drained in the spring and reflooded in the fall (seasonal). The prevalence of C. botulinum type C declined in seasonal marshes during the dry period. Similar declines did not occur in the permanently flooded marshes.

  10. Investigating CRISPR-Cas systems in Clostridium botulinum via bioinformatics tools.

    Science.gov (United States)

    Negahdaripour, Manica; Nezafat, Navid; Hajighahramani, Nasim; Rahmatabadi, Seyyed Soheil; Ghasemi, Younes

    2017-10-01

    The Clustered regularly interspaced short palindromic repeats (CRISPR) systems are a type of innate immunity found in some prokaryotes, which protect them against alien genetic elements by targeting foreign nucleic acids. Some other functions are also attributed to these systems. Clostridium botulinum bacteria produce botulinum neurotoxins (BoNT), one of the deadliest known toxins for humans and some animals. Food poisoning due to these bacteria is still a challenge in food industries. On the other hand, BoNT has been widely investigated for therapeutic applications including different muscle disorders. Bont genes may be located on bacterial chromosomes, plasmids, or even prophages. Generally, the genomes of Cl. botulinum show a high level of plasticity. In order to investigate the presence and characteristics of CRISPRs in these anaerobe bacteria, an in silico study on 113 CRISPR arrays identified in 38 Cl. botulinum strains was performed. A high occurrence of CRISPR arrays (80%) were found, with a remarkable frequency on plasmids. Several (CRISPR-associated) Cas proteins from different types were recognized in the studied strains, which were mostly Cas6. The CRISPR-Cas systems were identified as type I or III, but no type II. The spacers showed more homology with bacterial plasmids than phages. Active CRISPR-Cas systems can prevent the transfer of foreign genes, which may also include bont genes. This study provides the first insight into the probable roles of CRISPR-Cas systems in Cl. botulinum strains such as toxigenicity. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Clostridium botulinum group III: a group with dual identity shaped by plasmids, phages and mobile elements

    Directory of Open Access Journals (Sweden)

    Håfström Therese

    2011-04-01

    Full Text Available Abstract Background Clostridium botulinum strains can be divided into four physiological groups that are sufficiently diverged to be considered as separate species. Here we present the first complete genome of a C. botulinum strain from physiological group III, causing animal botulism. We also compare the sequence to three new draft genomes from the same physiological group. Results The 2.77 Mb chromosome was highly conserved between the isolates and also closely related to that of C. novyi. However, the sequence was very different from the human C. botulinum group genomes. Replication-directed translocations were rare and conservation of synteny was high. The largest difference between C. botulinum group III isolates occurred within their surprisingly large plasmidomes and in the pattern of mobile elements insertions. Five plasmids, constituting 13.5% of the total genetic material, were present in the completed genome. Interestingly, the set of plasmids differed compared to other isolates. The largest plasmid, the botulinum-neurotoxin carrying prophage, was conserved at a level similar to that of the chromosome while the medium-sized plasmids seemed to be undergoing faster genetic drift. These plasmids also contained more mobile elements than other replicons. Several toxins and resistance genes were identified, many of which were located on the plasmids. Conclusions The completion of the genome of C. botulinum group III has revealed it to be a genome with dual identity. It belongs to the pathogenic species C. botulinum, but as a genotypic species it should also include C. novyi and C. haemolyticum. The genotypic species share a conserved chromosomal core that can be transformed into various pathogenic variants by modulation of the highly plastic plasmidome.

  12. Clostridium botulinum group III: a group with dual identity shaped by plasmids, phages and mobile elements.

    Science.gov (United States)

    Skarin, Hanna; Håfström, Therese; Westerberg, Josefina; Segerman, Bo

    2011-04-12

    Clostridium botulinum strains can be divided into four physiological groups that are sufficiently diverged to be considered as separate species. Here we present the first complete genome of a C. botulinum strain from physiological group III, causing animal botulism. We also compare the sequence to three new draft genomes from the same physiological group. The 2.77 Mb chromosome was highly conserved between the isolates and also closely related to that of C. novyi. However, the sequence was very different from the human C. botulinum group genomes. Replication-directed translocations were rare and conservation of synteny was high. The largest difference between C. botulinum group III isolates occurred within their surprisingly large plasmidomes and in the pattern of mobile elements insertions. Five plasmids, constituting 13.5% of the total genetic material, were present in the completed genome. Interestingly, the set of plasmids differed compared to other isolates. The largest plasmid, the botulinum-neurotoxin carrying prophage, was conserved at a level similar to that of the chromosome while the medium-sized plasmids seemed to be undergoing faster genetic drift. These plasmids also contained more mobile elements than other replicons. Several toxins and resistance genes were identified, many of which were located on the plasmids. The completion of the genome of C. botulinum group III has revealed it to be a genome with dual identity. It belongs to the pathogenic species C. botulinum, but as a genotypic species it should also include C. novyi and C. haemolyticum. The genotypic species share a conserved chromosomal core that can be transformed into various pathogenic variants by modulation of the highly plastic plasmidome.

  13. Detection of Clostridium botulinum in liquid manure and biogas plant wastes.

    Science.gov (United States)

    Neuhaus, Jürgen; Schrödl, Wieland; Shehata, Awad A; Krüger, Monika

    2015-09-01

    Biogas plants have been considered as a source for possible amplification and distribution of pathogenic bacteria capable of causing severe infections in humans and animals. Manure and biogas wastes could be sources for spore-forming bacteria such as Clostridium botulinum. In the present study, 24 liquid manure and 84 biogas waste samples from dairies where the majority of the cows suffered from chronic botulism were investigated for the presence of botulinum neurotoxins (BoNT) and C. botulinum spores. The prevalence of BoNT/A, B, C, D, and E in biogas wastes was 16.6, 8.3, 10.7, 7.1, and 10.8 %, respectively, while in manure, the prevalence was 0.0, 0.0, 0.0, 8.3, and 4.1 %, respectively. After enrichment of samples in reinforced cultural medium, they were tested for C. botulinum BoNT/A, B, C, D, and E using ELISA (indirect C. botulinum detection). The prevalence of C. botulinum type A, B, C, D, and E samples in biogas wastes was 20.2, 15.5, 19, 10.7, and 34.8 %, respectively, while the prevalence in liquid manure was 0.0, 0.0, 0.0, 8.3, and 12.5 %, respectively. In conclusion, the occurrence of BoNT and C. botulinum spores in biogas waste of diseased animals indicates an increased and underestimated hygienic risk. Application of digestates from biogas fermentations as fertilizers could lead to an accumulation of long lifespan spores in the environment and could be a possible health hazard.

  14. Optimisation of protocol for Clostridium botulinum detection in mink feed

    Directory of Open Access Journals (Sweden)

    Grenda Tomasz

    2015-09-01

    Full Text Available As the test material mink feed with natural microflora was used. The analyses were conducted using Wrzosek and TPGY broth media, and Willis–Hobbs and Zeissler differential agar media. Wrzosek, Willis–Hobbs, and Zeissler media are described in Polish Standards approved by the National Standards Body in Poland and routinely used in detection of anaerobic bacteria in Poland. Detection and identification of C. botulinum was performed with a previously validated real-time PCR method based on ntnh gene detection, which is common in all C. botulinum toxotypes. The use of Wrzosek broth and Zeissler agar in routine analyses for detection and identification of C. botulinum was ineffective and limited. The obtained results showed the highest culturing process effectiveness in TPGY broth with 72 h incubation at 30°C and isolation on Willis–Hobbs agar. The real-time PCR method based on ntnh gene detection used in this study could be utilised as a supplementary tool to the mouse lethality assay.

  15. Meta-analysis of D-values of proteolytic Clostridium botulinum and its surrogate strain Clostridium sporogenes PA 3679.

    Science.gov (United States)

    Diao, Mamadou Moctar; André, Stéphane; Membré, Jeanne-Marie

    2014-03-17

    Foodborne botulism is a serious disease resulting from ingestion of preformed Clostridium botulinum neurotoxin in foodstuff. Since the 19th century, the heat resistance of this spore forming bacteria has been extensively studied in order to guarantee the public health associated with low acidic, ambient stable products. The most largely used heat resistance parameters in thermal settings of such products are the D121.1°C values (time required to have a 10-fold decrease of the spore count, at 121.1°C) and the z-values (temperature increase to have a 10-fold decrease of D-values). To determine D121.1°C and z-values of proteolytic C. botulinum and its nontoxigenic surrogate strain C. sporogenes PA3679, a dataset of 911 D-values was collected from 38 scientific studies. Within a meta-analysis framework, a mixed-effect linear model was developed with the log D-value (min) as response and the heat treatment temperature as explicative variable. The studies (38), the C. botulinum strains (11), and the heat treatment media (liquid media and various food matrices, split into nine categories in total) were considered as co-variables having a random effect. The species (C. botulinum and C. sporogenes) and the pH (five categories) were considered as co-variables having a fixed effect. Overall, the model gave satisfactory results with a residual standard deviation of 0.22. The heat resistance of proteolytic C. botulinum was found significantly lower than the C. sporogenes PA 3679 one: the mean D-values at the reference temperature of 121.1°C, in liquid media and pH neutral, were estimated to 0.19 and 1.28min for C. botulinum and C. sporogenes, respectively. On the other hand, the mean z-values of the two species were similar: 11.3 and 11.1°C for C. botulinum and C. sporogenes, respectively. These results will be applied to thermal settings of low-acid ambient stable products. Copyright © 2014. Published by Elsevier B.V.

  16. Attempts to identify Clostridium botulinum toxin in milk from three experimentally intoxicated Holstein cows

    Science.gov (United States)

    Moeller, R.B.; Puschner, B.; Walker, R.L.; Rocke, T.E.; Smith, S.R.; Cullor, J.S.; Ardans, A.A.

    2009-01-01

    Three adult lactating Holstein cows were injected in the subcutaneous abdominal vein with 175 ng/kg of body weight of Clostridium botulinum type C toxin (451 cow median toxic doses) to determine if this botulinum toxin crosses the blood–milk barrier. Whole blood (in sodium heparin) and clotted blood serum samples were taken at 0 min, 10 min, and 3, 6, 9, and 12 h postinoculation. Milk samples were taken at 0 min and at 3, 6, 9 and 12 h postinoculation. All samples were tested for the presence of the toxin using the mouse bioassay and immunostick ELISA test. The immunostick ELISA identified the toxin in whole blood and the mouse bioassay identified the toxin in serum at all times examined in all 3 animals. Toxin was not identified by either detection method in milk samples collected from the 3 animals. From these results, it appears that Clostridium botulinum type C toxin does not cross from the blood to the milk in detectable concentrations.

  17. Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum

    Directory of Open Access Journals (Sweden)

    Twine Susan M

    2009-03-01

    Full Text Available Abstract Background Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Results Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes, and the flagellar glycosylation island (FGI. These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5 has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Conclusion Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism

  18. An atypical outbreak of food-borne botulism due to Clostridium botulinum types B and E from ham.

    Science.gov (United States)

    Mazuet, Christelle; Sautereau, Jean; Legeay, Christine; Bouchier, Christiane; Bouvet, Philippe; Popoff, Michel R

    2015-02-01

    An outbreak of human botulism was due to consumption of ham containing botulinum neurotoxins B and E. A Clostridium botulinum type E strain isolated from ham was assigned to a new subtype (E12) based on bont/E gene sequencing and belongs to a new multilocus sequence subtype, as analyzed by whole-genome sequencing. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Transcriptomic analysis of (group I Clostridium botulinum ATCC 3502 cold shock response.

    Directory of Open Access Journals (Sweden)

    Elias Dahlsten

    Full Text Available Profound understanding of the mechanisms foodborne pathogenic bacteria utilize in adaptation to the environmental stress they encounter during food processing and storage is of paramount importance in design of control measures. Chill temperature is a central control measure applied in minimally processed foods; however, data on the mechanisms the foodborne pathogen Clostridium botulinum activates upon cold stress are scarce. Transcriptomic analysis on the C. botulinum ATCC 3502 strain upon temperature downshift from 37°C to 15°C was performed to identify the cold-responsive gene set of this organism. Significant up- or down-regulation of 16 and 11 genes, respectively, was observed 1 h after the cold shock. At 5 h after the temperature downshift, 199 and 210 genes were up- or down-regulated, respectively. Thus, the relatively small gene set affected initially indicated a targeted acute response to cold shock, whereas extensive metabolic remodeling appeared to take place after prolonged exposure to cold. Genes related to fatty acid biosynthesis, oxidative stress response, and iron uptake and storage were induced, in addition to mechanisms previously characterized as cold-tolerance related in bacteria. Furthermore, several uncharacterized DNA-binding transcriptional regulator-encoding genes were induced, suggesting involvement of novel regulatory mechanisms in the cold shock response of C. botulinum. The role of such regulators, CBO0477 and CBO0558A, in cold tolerance of C. botulinum ATCC 3502 was demonstrated by deteriorated growth of related mutants at 17°C.

  20. Use of gamma radiation on control of Clostridium botulinum in mortadella formulated with different nitrite levels

    Science.gov (United States)

    Dutra, Monalisa Pereira; Aleixo, Glécia de Cássia; Ramos, Alcinéia de Lemos Souza; Silva, Maurício Henriques Louzada; Pereira, Marcio Tadeu; Piccoli, Roberta Hilsdorf; Ramos, Eduardo Mendes

    2016-02-01

    This study investigated the effects of applying different doses of gamma radiation (0, 10 and 20 kGy) on Clostridium botulinum spores (107 spores/g) inoculated into mortadellas with different nitrite contents (0, 150 and 300 ppm). We also evaluated the order of application of heat (cooking) and irradiation processing. The products were evaluated for survival of C. botulinum, pH, water activity (Aw), redox potential (Eh) and residual nitrite content. In the non-irradiated raw batters, almost all spores could be recovered when no nitrite was added and only half was recovered with the addition of 150 ppm of nitrite. The use of 150 ppm of nitrite was able to inhibit the germination or growth of C. botulinum in non-irradiated cooked mortadellas after 48 h of processing. However, after 30 days of chilling storage (4 °C), it was possible to recover 105 UFC/g of this microorganism. The gamma irradiation (>10 kGy) had a positive effect on the inactivation of C. botulinum in mortadellas, independent of the sodium nitrite level used and the cooking/irradiation processing order.

  1. Different substrate recognition requirements for cleavage of synaptobrevin-2 by Clostridium baratii and Clostridium botulinum type F neurotoxins.

    Science.gov (United States)

    Kalb, Suzanne R; Baudys, Jakub; Egan, Christina; Smith, Theresa J; Smith, Leonard A; Pirkle, James L; Barr, John R

    2011-02-01

    Botulinum neurotoxins (BoNTs) cause botulism, which can be fatal if it is untreated. BoNTs cleave proteins necessary for nerve transmission, resulting in paralysis. The in vivo protein target has been reported for all seven serotypes of BoNT, i.e., serotypes A to G. Knowledge of the cleavage sites has led to the development of several assays to detect BoNT based on its ability to cleave a peptide substrate derived from its in vivo protein target. Most serotypes of BoNT can be subdivided into subtypes, and previously, we demonstrated that three of the currently known subtypes of BoNT/F cleave a peptide substrate, a shortened version of synaptobrevin-2, between Q58 and K59. However, our research indicated that Clostridium baratii type F toxin did not cleave this peptide. In this study, we detail experiments demonstrating that Clostridium baratii type F toxin cleaves recombinant synaptobrevin-2 in the same location as that cleaved by proteolytic F toxin. In addition, we demonstrate that Clostridium baratii type F toxin can cleave a peptide substrate based on the sequence of synaptobrevin-2. This peptide substrate is an N-terminal extension of the original peptide substrate used for detection of other BoNT/F toxins and can be used to detect four of the currently known BoNT/F subtypes by mass spectrometry.

  2. Regulation of Botulinum Neurotoxin Synthesis and Toxin Complex Formation by Arginine and Glucose in Clostridium botulinum ATCC 3502.

    Science.gov (United States)

    Fredrick, Chase M; Lin, Guangyun; Johnson, Eric A

    2017-07-01

    Botulinum neurotoxin (BoNT), produced by neurotoxigenic clostridia, is the most potent biological toxin known and the causative agent of the paralytic disease botulism. The nutritional, environmental, and genetic regulation of BoNT synthesis, activation, stability, and toxin complex (TC) formation is not well studied. Previous studies indicated that growth and BoNT formation were affected by arginine and glucose in Clostridium botulinum types A and B. In the present study, C. botulinum ATCC 3502 was grown in toxin production medium (TPM) with different levels of arginine and glucose and of three products of arginine metabolism, citrulline, proline, and ornithine. Cultures were analyzed for growth (optical density at 600 nm [OD 600 ]), spore formation, and BoNT and TC formation by Western blotting and immunoprecipitation and for BoNT activity by mouse bioassay. A high level of arginine (20 g/liter) repressed BoNT production approximately 1,000-fold, enhanced growth, slowed lysis, and reduced endospore production by greater than 1,000-fold. Similar effects on toxin production were seen with equivalent levels of citrulline but not ornithine or proline. In TPM lacking glucose, levels of formation of BoNT/A1 and TC were significantly decreased, and extracellular BoNT and TC proteins were partially inactivated after the first day of culture. An understanding of the regulation of C. botulinum growth and BoNT and TC formation should be valuable in defining requirements for BoNT formation in foods and clinical samples, improving the quality of BoNT for pharmaceutical preparations, and elucidating the biological functions of BoNTs for the bacterium. IMPORTANCE Botulinum neurotoxin (BoNT) is a major food safety and bioterrorism concern and is also an important pharmaceutical, and yet the regulation of its synthesis, activation, and stability in culture media, foods, and clinical samples is not well understood. This paper provides insights into the effects of critical

  3. Association of toxin-producing Clostridium botulinum with the macroalga Cladophora in the Great Lakes

    Science.gov (United States)

    Chun, Chan Lan; Ochsner, Urs; Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Tepp, William H.; Lin, Guangyun; Johnson, Eric A.; Peller, Julie; Sadowsky, Michael J.

    2013-01-01

    Avian botulism, a paralytic disease of birds, often occurs on a yearly cycle and is increasingly becoming more common in the Great Lakes. Outbreaks are caused by bird ingestion of neurotoxins produced by Clostridium botulinum, a spore-forming, gram-positive, anaerobe. The nuisance, macrophytic, green alga Cladophora (Chlorophyta; mostly Cladophora glomerata L.) is a potential habitat for the growth of C. botulinum. A high incidence of botulism in shoreline birds at Sleeping Bear Dunes National Lakeshore (SLBE) in Lake Michigan coincides with increasingly massive accumulations of Cladophora in nearshore waters. In this study, free-floating algal mats were collected from SLBE and other shorelines of the Great Lakes between June and October 2011. The abundance of C. botulinum in algal mats was quantified and the type of botulism neurotoxin (bont) genes associated with this organism were determined by using most-probable-number PCR (MPN-PCR) and five distinct bont gene-specific primers (A, B, C, E, and F). The MPN-PCR results showed that 16 of 22 (73%) algal mats from the SLBE and 23 of 31(74%) algal mats from other shorelines of the Great Lakes contained the bont type E (bont/E) gene. C. botulinum was present up to 15 000 MPN per gram dried algae based on gene copies of bont/E. In addition, genes for bont/A and bont/B, which are commonly associated with human diseases, were detected in a few algal samples. Moreover, C. botulinum was present as vegetative cells rather than as dormant spores in Cladophora mats. Mouse toxin assays done using supernatants from enrichment of Cladophora containing high densities of C. botulinum (>1000 MPN/g dried algae) showed that Cladophora-borne C. botulinum were toxin-producing species (BoNT/E). Our results indicate that Cladophora provides a habitat for C. botulinum, warranting additional studies to better understand the relationship between this bacterium and the alga, and how this interaction potentially contributes to botulism

  4. Proposed role of lactate in germination of hypochlorite-treated Clostridium botulinum spores.

    OpenAIRE

    Foegeding, P M; Busta, F F

    1983-01-01

    Clostridium botulinum 12885A spores treated with hypochlorite required added DL-calcium lactate for L-alanine germination. Lactate was the active component of calcium lactate. Equimolar concentrations of L-malate, but not of DL-propionate, could replace lactate, suggesting that the alpha-hydroxy acid structure is important. Neither lactate nor malate was an effective germinant for buffer-treated or hypochlorite-treated spores. If the L-alanine concentration was increased 100-fold (to 450 mM),...

  5. Complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in Clostridium botulinum type B strain 111 isolated from an infant patient in Japan.

    Science.gov (United States)

    Hosomi, Koji; Sakaguchi, Yoshihiko; Kohda, Tomoko; Gotoh, Kazuyoshi; Motooka, Daisuke; Nakamura, Shota; Umeda, Kaoru; Iida, Tetsuya; Kozaki, Shunji; Mukamoto, Masafumi

    2014-12-01

    Botulinum neurotoxins (BoNTs) are highly potent toxins that are produced by Clostridium botulinum. We determined the complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in C. botulinum type B strain 111 in order to obtain an insight into the toxigenicity and evolution of the bont gene in C. botulinum. Group I C. botulinum type B strain 111 was isolated from the first case of infant botulism in Japan in 1995. In previous studies, botulinum neurotoxin subtype B2 (BoNT/B2) produced by strain 111 exhibited different antigenic properties from those of authentic BoNT/B1 produced by strain Okra. We have recently shown that the isolates of strain 111 that lost toxigenicity were cured of the plasmid containing the bont/B2 gene. In the present study, the plasmid (named pCB111) was circular 265,575 bp double-stranded DNA and contained 332 predicted open reading frames (ORFs). 85 gene products of these ORFs could be functionally assigned on the basis of sequence homology to known proteins. The bont/B2 complex genes were located on pCB111 and some gene products may be involved in the conjugative plasmid transfer and horizontal transfer of bont genes. pCB111 was similar to previously identified plasmids containing bont/B1, /B5, or/A3 complex genes in other group I C. botulinum strains. It was suggested that these plasmids had been derived from a common ancestor and had played important roles for the bont gene transfer between C. botulinum.

  6. Study on PotentialClostridium BotulinumGrowth and Toxin Production in Parma Ham.

    Science.gov (United States)

    Merialdi, Giuseppe; Ramini, Mattia; Parolari, Giovanni; Barbuti, Silvana; Frustoli, Maria Angela; Taddei, Roberta; Pongolini, Stefano; Ardigò, Paolo; Cozzolino, Paolo

    2016-04-19

    The objective of this study was to investigate Clostridium botulinum growth and toxin production in the industrially manufactured Italian Parma ham. The study focuses on the Parma ham production phase identified as maximum risk to C. botulinum proliferation, i.e . the transition from cold phase (salting and resting) to a phase carried out at temperature between 15 and 23°C (drying). A preliminary in vitro test was carried out in order to verify the capability of 6 C. botulinum strains (1 type A, 4 type B, and 1 type E strains) to grow in conditions of temperature, pH and NaCl concentration comparable to those of the beginning stage of ham drying. Five C. botulinum strains grew at 20°C and pH 6, four strains produced toxin when inoculated at a concentration equal to 10 3 cfu/mL at NaCl concentration of 4%, while when the inoculum concentration was 10 cfu/mL, NaCl concentration of 3% resulted the toxin-genesis limiting factor. An experimental contamination with a mixture of the 5 C. botulinum strains selected by the preliminary in vitro test was performed on 9 thighs inoculated at the end of the resting phase. The study was designed to evaluate the potential growth and toxin production in extremely favourable conditions for the bacterium. Type B proteolytic C. botulinum toxin was produced after 14 days of incubation at 20°C in 2 thighs characterised by high weight, low number of days of resting and anomalous physiochemical characteristics [one for very low NaCl concentration (1.59%), the other for elevated pH (6.27) and both for high water activity values (>0.970)]. The results of this research confirm that the cold resting step is a critical phase in the production process of Parma ham for the investigated hazard. Based on the present study, the long resting phase adopted in the manufacturing of Parma ham is proven effective to prevent the growth of C. botulinum , an event which could not otherwise be excluded if the hams were processed under less stringent

  7. Modification of an acetone-sodium dodecyl sulfate disruption method for cellular protein extraction from neuropathogenic Clostridium botulinum

    Science.gov (United States)

    An acetone-sodium dodecyl sulfate (SDS) disruption method was used for the extraction of cellular proteins from neurotoxigenic Clostridium botulinum. The amount of protein extracted per gram of dry weight and the protein profile as revealed by polyacrylamide gel electrophoresis (PAGE) was comparabl...

  8. BIODEGRADATION OF POLYBLEND POLYPROPILENE- PALM OIL-AMYLUM BY Bacillus subtilus AND Clostridium botulinum

    Directory of Open Access Journals (Sweden)

    Zainal Fanani

    2010-06-01

    Full Text Available It had been done a biodegradation polyblend from blending polypropilene-palm oil-amylum with three composition of polyblend i.e polyblend A 80% polypropilene- 19.5% palm oil- 0.5% amylum, poliblend B 80% polypropilene- 19% palm oil- 1% amylum and polyblend C 80% polypropilene-18% palm oil- 2% amylum by B. subtilus and C. botulinum, time incubation was twenty five days. The characterization of polyblend before and after biodegradation has done with FTIR, DTA, Viscometre and tensile strength of polyblend. The result showed that Bacillus subtilus and Clostridium botulinum can biodegradate polyblend and make holes as well as chink on polyblend especially polyblend C, because it has more carbohidrat than polyblend A and B. Analysis from FTIR showed compatible of poliblend because it did not have a new function group and did not change of wavelength. Data of tensile strength showed lower value after biodegradation at polyblend C and from DTA and Viscometre showed lower melting point and lower average molecule weight, respectively.   Keywords: Biodegradation, Polyblend, Bacillus, Clostridium

  9. New Insights into the genetic diversity of Clostridium botulinum Group III through extensive genome exploration

    Directory of Open Access Journals (Sweden)

    Cédric eWoudstra

    2016-05-01

    Full Text Available Animal botulism is caused by group III Clostridium botulinum strains producing type C and D toxins, or their chimeric forms C/D and D/C. Animal botulism is considered an emerging disease in Europe, notably in poultry production. Before our study, 14 genomes from different countries were available in the public database, but none were from France. In order to investigate the genetic relationship of French strains with different geographical areas and find new potential typing targets, 17 strains of C. botulinum group III were sequenced (16 from France and one from New Caledonia. Fourteen were type C/D strains isolated from chickens, ducks, guinea fowl and turkeys and three were type D/C strains isolated from cattle. The New Caledonian strain was a type D/C strain. Whole genome sequence analysis showed the French strains to be closely related to European strains from C. botulinum group III lineages Ia and Ib. The investigation of CRISPR sequences as genetic targets for differentiating strains in group III proved to be irrelevant for type C/D due to a deficient CRISPR/Cas mechanism, but not for type D/C. Conversely, the extrachromosomal elements of type C/D strains could be used to generate a genetic ID card. The highest level of discrimination was achieved with SNP core phylogeny, which allowed differentiation up to strain level and provide the most relevant information for genetic epidemiology studies and discrimination.

  10. Molecular Assembly of Clostridium botulinum progenitor M complex of type E.

    Science.gov (United States)

    Eswaramoorthy, Subramaniam; Sun, Jingchuan; Li, Huilin; Singh, Bal Ram; Swaminathan, Subramanyam

    2015-12-07

    Clostridium botulinum neurotoxin (BoNT) is released as a progenitor complex, in association with a non-toxic-non-hemagglutinin protein (NTNH) and other associated proteins. We have determined the crystal structure of M type Progenitor complex of botulinum neurotoxin E [PTC-E(M)], a heterodimer of BoNT and NTNH. The crystal structure reveals that the complex exists as a tight, interlocked heterodimer of BoNT and NTNH. The crystal structure explains the mechanism of molecular assembly of the complex and reveals several acidic clusters at the interface responsible for association at low acidic pH and disassociation at basic/neutral pH. The similarity of the general architecture between the PTC-E(M) and the previously determined PTC-A(M) strongly suggests that the progenitor M complexes of all botulinum serotypes may have similar molecular arrangement, although the neurotoxins apparently can take very different conformation when they are released from the M complex.

  11. The purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Clostridium botulinum

    Energy Technology Data Exchange (ETDEWEB)

    Dobson, Renwick C. J., E-mail: rdobson@unimelb.edu.au; Atkinson, Sarah C. [Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Parkville, Victoria 3010 (Australia); Gorman, Michael A. [St Vincents Institute, 9 Princes Street, Fitzroy, Victoria 3065 (Australia); Newman, Janet M. [CSIRO Division of Molecular and Health Technologies, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Parker, Michael W. [Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Parkville, Victoria 3010 (Australia); St Vincents Institute, 9 Princes Street, Fitzroy, Victoria 3065 (Australia); Perugini, Matthew A. [Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Parkville, Victoria 3010 (Australia)

    2008-03-01

    Dihydrodipicolinate synthase (DHDPS), an enzyme in the lysine-biosynthetic pathway, is a promising target for antibiotic development against pathogenic bacteria. Here, the expression, purification, crystallization and preliminary diffraction analysis of DHDPS from C. botulinum are reported. In recent years, dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) has received considerable attention from both mechanistic and structural viewpoints. This enzyme, which is part of the diaminopimelate pathway leading to lysine, couples (S)-aspartate-β-semialdehyde with pyruvate via a Schiff base to a conserved active-site lysine. In this paper, the expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPS from Clostridium botulinum, an important bacterial pathogen, are presented. The enzyme was crystallized in a number of forms, predominantly using PEG precipitants, with the best crystal diffracting to beyond 1.9 Å resolution and displaying P4{sub 2}2{sub 1}2 symmetry. The unit-cell parameters were a = b = 92.9, c = 60.4 Å. The crystal volume per protein weight (V{sub M}) was 2.07 Å{sup 3} Da{sup −1}, with an estimated solvent content of 41%. The structure of the enzyme will help guide the design of novel therapeutics against the C. botulinum pathogen.

  12. High pressure inactivation of Clostridium botulinum type E endospores in model emulsion systems

    Science.gov (United States)

    Schnabel, Juliane; Lenz, Christian A.; Vogel, Rudi F.

    2015-01-01

    Clostridium botulinum type E is a cold-tolerant, neurotoxigenic, endospore-forming organism, primarily associated with aquatic environments. High pressure thermal (HPT) processing presents a promising tool to enhance food safety and stability. The effect of fat on HPT inactivation of C. botulinum type E spores was investigated using an emulsion model system. The distribution of spores in oil-in-water (O/W) emulsions and their HPT (300-750 MPa, 45-75 °C, 10 min) inactivation was determined as a function of emulsion fat content (30-70% (v/v) soybean oil in buffer). Approximately 26% and 74% of the spores were located at the oil-buffer interface and the continuous phase, respectively. Spore inactivation in emulsion systems decreased with increasing oil contents, which suggests that the fat content of food plays an important role in the protection of C. botulinum type E endospores against HPT treatments. These results can be helpful for future safety considerations. This paper was presented at the 8th International Conference on High Pressure Bioscience & Biotechnology (HPBB 2014), in Nantes (France), 15-18 July 2014.

  13. Occurrence of Clostridium botulinum neurotoxin in chronic disease of dairy cows.

    Science.gov (United States)

    Seyboldt, Christian; Discher, Sabrina; Jordan, Eva; Neubauer, Heinrich; Jensen, Katharina Charlotte; Campe, Amely; Kreienbrock, Lothar; Scheu, Theresa; Wichern, Anika; Gundling, Frieder; DoDuc, Phuong; Fohler, Svenja; Abdulmawjood, Amir; Klein, Günter; Hoedemaker, Martina

    2015-06-12

    Botulism caused by neurotoxins of Clostridium (C.) botulinum is a rare, but serious life-threatening disease in humans and animals. Botulism in livestock is usually caused by the oral uptake of C. botulinum neurotoxins (BoNT) via contaminated feed and is characterized by flaccid paralysis. In the recent past a new syndrome caused by BoNT in dairy cattle was postulated. It was supposed that C. botulinum is able to colonize the lower intestine and may subsequently produce neurotoxin. The continuous resorption of small amounts of these BoNT may then provoke the so called syndrome of "chronic" or "visceral" botulism involving unspecific clinical symptoms, reduced performance of dairy cows and massive animal losses in the affected herd. To test this hypothesis a case-control study was conducted involving 92 affected farms and 47 control farms located in Northern Germany. Fecal samples of 1388 animals were investigated for the presence of BoNT to verify the key requirement of the hypothesis of chronic botulism. BoNT was not detected in any of the fecal samples using the most sensitive standard method for BoNT detection, the mouse bioassay. Therefore, the existence of "chronic" or "visceral" botulism could not be proven. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  14. Distinguishing highly-related outbreak-associated Clostridium botulinum type A(B) strains.

    Science.gov (United States)

    Raphael, Brian H; Shirey, Timothy B; Lúquez, Carolina; Maslanka, Susan E

    2014-07-16

    In the United States, most Clostridium botulinum type A strains isolated during laboratory investigations of human botulism demonstrate the presence of an expressed type A botulinum neurotoxin (BoNT/A) gene and an unexpressed BoNT/B gene. These strains are designated type A(B). The most common pulsed-field gel electrophoresis (PFGE) pattern in the C. botulinum PulseNet database is composed of A(B) strains. The purpose of this study was to evaluate the ability of genome sequencing and multi-loci variable number of tandem repeat analysis (MLVA) to differentiate such strains. The genome sequences of type A(B) strains evaluated in this study are closely related and cluster together compared to other available C. botulinum Group I genomes. In silico multilocus sequence typing (MLST) analysis (7-loci) was unable to differentiate any of the type A(B) strains isolated from seven different outbreak investigations evaluated in this study. A 15-locus MLVA scheme demonstrated an improved ability to differentiate these strains, however, repeat unit variation among the strains was restricted to only two loci. Reference-free single nucleotide polymorphism (SNP) analysis demonstrated the ability to differentiate strains from all of the outbreaks examined and a non-outbreak associated strain. This study confirms that type A(B) strains that share the same PFGE pattern also share closely-related genome sequences. The lack of a complete type A(B) strain representative genome sequence hinders the ability to assemble genomes by reference mapping and analysis of SNPs at pre-identified sites. However, compared to other methods evaluated in this study, a reference-free SNP analysis demonstrated optimal subtyping utility for type A(B) strains using de novo assembled genome sequences.

  15. Structure- and Substrate- Based Inhibitor Design for Clostridium botulinum Neurotoxin Serotype A*

    Energy Technology Data Exchange (ETDEWEB)

    Kumaran,D.; Rawat, R.; Ludivico, M.; Ahmed, S.; Swaminathan, S.

    2008-01-01

    The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins cleave specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex proteins and block the release of neurotransmitters that cause flaccid paralysis and are considered potential bioweapons. Botulinum neurotoxin type A is the most potent among the clostridial neurotoxins, and to date there is no post-exposure therapeutic intervention available. To develop inhibitors leading to drug design, it is imperative that critical interactions between the enzyme and the substrate near the active site are known. Although enzyme-substrate interactions at exosites away from the active site are mapped in detail for botulinum neurotoxin type A, information about the active site interactions is lacking. Here, we present the crystal structures of botulinum neurotoxin type A catalytic domain in complex with four inhibitory substrate analog tetrapeptides, viz. RRGC, RRGL, RRGI, and RRGM at resolutions of 1.6-1.8 Angstroms . These structures show for the first time the interactions between the substrate and enzyme at the active site and delineate residues important for substrate stabilization and catalytic activity. We show that OH of Tyr366 and NH2 of Arg363 are hydrogen-bonded to carbonyl oxygens of P1 and P1' of the substrate analog and position it for catalytic activity. Most importantly, the nucleophilic water is replaced by the amino group of the N-terminal residue of the tetrapeptide. Furthermore, the S1' site is formed by Phe194, Thr215, Thr220, Asp370, and Arg363. The Ki of the best inhibitory tetrapeptide is 157 nm.

  16. Rapid Detection of Clostridium botulinum Toxins A, B, E, and F in Clinical Samples, Selected Food Matrices, and Buffer Using Paramagnetic Bead-Based Electrochemiluminescence Detection

    National Research Council Canada - National Science Library

    Rivera, Victor R; Gamez, Frank J; Keener, William K; White, Jill A; Poli, Mark A

    2006-01-01

    Sensitive and specific electrochemiluminescence (ECL) assays were used to detect Clostridium botulinum neurotoxins serotypes A, B, E, and F in undiluted human serum, undiluted human urine, assay buffer, and selected food matrices...

  17. Prevalence of toxin-producing Clostridium botulinum associated with the macroalga Cladophora in three Great Lakes: growth and management

    Science.gov (United States)

    Chun, Chan Lan; Kahn, Chase I.; Borchert, Andrew J.; Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Peller, Julie R.; Pier, Christina; Lin, Guangyun; Johnson, Eric A.; Sadowsky, Michael J.

    2015-01-01

    The reemergence of avian botulism caused by Clostridium botulinum type E has been observed across the Great Lakes in recent years. Evidence suggests an association between the nuisance algae, Cladophoraspp., and C. botulinum in nearshore areas of the Great Lakes. However, the nature of the association between Cladophora and C. botulinum is not fully understood due, in part, to the complex food web interactions in this disease etiology. In this study, we extensively evaluated their association by quantitatively examining population size and serotypes of C. botulinum in algal mats collected from wide geographic areas in lakes Michigan, Ontario, and Erie in 2011–2012 and comparing them with frequencies in other matrices such as sand and water. A high prevalence (96%) of C. botulinum type E was observed inCladophora mats collected from shorelines of the Great Lakes in 2012. Among the algae samples containing detectable C. botulinum, the population size of C. Botulinum type E was 100–104 MPN/g dried algae, which was much greater (up to 103 fold) than that found in sand or the water column, indicating thatCladophora mats are sources of this pathogen. Mouse toxinantitoxin bioassays confirmed that the putativeC. botulinum belonged to the type E serotype. Steam treatment was effective in reducing or eliminating C. botulinum type E viable cells in Cladophora mats, thereby breaking the potential transmission route of toxin up to the food chain. Consequently, our data suggest that steam treatment incorporated with a beach cleaning machine may be an effective treatment of Cladophora-borne C. botulinum and may reduce bird mortality and human health risks.

  18. A penicillin- and metronidazole-resistant Clostridium botulinum strain responsible for an infant botulism case.

    Science.gov (United States)

    Mazuet, C; Yoon, E-J; Boyer, S; Pignier, S; Blanc, T; Doehring, I; Meziane-Cherif, D; Dumant-Forest, C; Sautereau, J; Legeay, C; Bouvet, P; Bouchier, C; Quijano-Roy, S; Pestel-Caron, M; Courvalin, P; Popoff, M R

    2016-07-01

    The clinical course of a case of infant botulism was characterized by several relapses despite therapy with amoxicillin and metronidazole. Botulism was confirmed by identification of botulinum toxin and Clostridium botulinum in stools. A C. botulinum A2 strain resistant to penicillins and with heterogeneous resistance to metronidazole was isolated from stool samples up to 110 days after onset. Antibiotic susceptibility was tested by disc agar diffusion and MICs were determined by Etest. Whole genome sequencing allowed detection of a gene cluster composed of blaCBP for a novel penicillinase, blaI for a regulator, and blaR1 for a membrane-bound penicillin receptor in the chromosome of the C. botulinum isolate. The purified recombinant penicillinase was assayed. Resistance to β-lactams was in agreement with the kinetic parameters of the enzyme. In addition, the β-lactamase gene cluster was found in three C. botulinum genomes in databanks and in two of 62 genomes of our collection, all the strains belonging to group I C. botulinum. This is the first report of a C. botulinum isolate resistant to penicillins. This stresses the importance of antibiotic susceptibility testing for adequate therapy of botulism. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  19. Neurotoxin synthesis is positively regulated by the sporulation transcription factor Spo0A in Clostridium botulinum type E.

    Science.gov (United States)

    Mascher, Gerald; Mertaoja, Anna; Korkeala, Hannu; Lindström, Miia

    2017-10-01

    Clostridium botulinum produces the most potent natural toxin, the botulinum neurotoxin (BoNT), probably to create anaerobiosis and nutrients by killing the host, and forms endospores that facilitate survival in harsh conditions and transmission. Peak BoNT production coincides with initiation of sporulation in C. botulinum cultures, which suggests common regulation. Here, we show that Spo0A, the master regulator of sporulation, positively regulates BoNT production. Insertional inactivation of spo0A in C. botulinum type E strain Beluga resulted in significantly reduced BoNT production and in abolished or highly reduced sporulation in relation to wild-type controls. Complementation with spo0A restored BoNT production and sporulation. Recombinant DNA-binding domain of Spo0A directly bound to a putative Spo0A-binding box (CTTCGAA) within the BoNT/E operon promoter, demonstrating direct regulation. Spo0A is the first neurotoxin regulator reported in C. botulinum type E. Unlike other C. botulinum strains that are terrestrial and employ the alternative sigma factor BotR in directing BoNT expression, C. botulinum type E strains are adapted to aquatic ecosystems, possess distinct epidemiology and lack BotR. Our results provide fundamental new knowledge on the genetic control of BoNT production and demonstrate common regulation of BoNT production and sporulation, providing a key intervention point for control. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  20. Development and characterization of six monoclonal antibodies to hemagglutinin-70 (HA70) of Clostridium botulinum and their application in a sandwich ELISA

    Science.gov (United States)

    Botulinum neurotoxins (BoNT),produced by Clostridium botulinum, cause severe neuroparalytic disease that if not treated quickly is often fatal. The toxin is synthesized as a 150 kDa precursor protein (holotoxin), which is then enzymatically cleaved to form two subunit chains linked by a single disul...

  1. The susceptibility of the mallard duck (Anas platyrhynchos) to Clostridium botulinum C2 toxin

    Science.gov (United States)

    Jensen, Wayne I.; Duncan, Ruth M.

    1980-01-01

    Most strains of Clostridium botulinum type C, after having lost their capacity to produce their dominant toxin (C1) as a result of being“cured”of their prophages, continue to produce C2, a trypsin-activable toxin reported by other investigators. While of relatively low toxicity when administered perorally to the adult mallard duck (Anas platyrhynchos), it was highly toxic when given parenterally. By the intravenous route, for example, it was more than 1, 000 times as toxic as C1 toxin by the same route, when compared on the basis of mouse intraperitoneal toxicity. The cause of death in every instance was massive pulmonary edema and hemorrhage rather than the respiratory paralysis that occurs in C1 intoxication.抄録

  2. Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus

    International Nuclear Information System (INIS)

    Rose, S.A.; Bailey, N.E.; Stringer, M.F.; Modi, N.K.; Tranter, H.S.; Hambleton, P.

    1989-01-01

    The effects of irradiation of Clostridium botulinum neutotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16-26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation. (author)

  3. Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus

    International Nuclear Information System (INIS)

    Rose, S.A.; Bailey, N.E.; Stringer, M.F.; Modi, N.K.; Tranter, H.S.; Hambleton, P.

    1988-01-01

    The effects of irradiation of Clostridium botulinum neurotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16-26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation. (author)

  4. Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.

    Science.gov (United States)

    Amatsu, Sho; Sugawara, Yo; Matsumura, Takuhiro; Kitadokoro, Kengo; Fujinaga, Yukako

    2013-12-06

    Clostridium botulinum HA is a component of the large botulinum neurotoxin complex and is critical for its oral toxicity. HA plays multiple roles in toxin penetration in the gastrointestinal tract, including protection from the digestive environment, binding to the intestinal mucosal surface, and disruption of the epithelial barrier. At least two properties of HA contribute to these roles: the sugar-binding activity and the barrier-disrupting activity that depends on E-cadherin binding of HA. HA consists of three different proteins, HA1, HA2, and HA3, whose structures have been partially solved and are made up mainly of β-strands. Here, we demonstrate structural and functional reconstitution of whole HA and present the complete structure of HA of serotype B determined by x-ray crystallography at 3.5 Å resolution. This structure reveals whole HA to be a huge triskelion-shaped molecule. Our results suggest that whole HA is functionally and structurally separable into two parts: HA1, involved in recognition of cell-surface carbohydrates, and HA2-HA3, involved in paracellular barrier disruption by E-cadherin binding.

  5. Domain Organization in Clostridium botulinum Neurotoxin Type E is Unique: Its Implication in Faster Translocation

    Energy Technology Data Exchange (ETDEWEB)

    Kumaran, D.; Eswaramoorthy, S; Furey, W; Navaza, J; Sax, M; Swaminathan, S

    2009-01-01

    Clostridium botulinum produces seven antigenically distinct neurotoxins [C. botulinum neurotoxins (BoNTs) A-G] sharing a significant sequence homology. Based on sequence and functional similarity, it was believed that their three-dimensional structures will also be similar. Indeed, the crystal structures of BoNTs A and B exhibit similar fold and domain association where the translocation domain is flanked on either side by binding and catalytic domains. Here, we report the crystal structure of BoNT E holotoxin and show that the domain association is different and unique, although the individual domains are similar to those of BoNTs A and B. In BoNT E, both the binding domain and the catalytic domain are on the same side of the translocation domain, and all three have mutual interfaces. This unique association may have an effect on the rate of translocation, with the molecule strategically positioned in the vesicle for quick entry into cytosol. Botulism, the disease caused by BoNT E, sets in faster than any other serotype because of its speedy internalization and translocation, and the present structure offers a credible explanation. We propose that the translocation domain in other BoNTs follows a two-step process to attain translocation-competent conformation as in BoNT E. We also suggest that this translocation-competent conformation in BoNT E is a probable reason for its faster toxic rate compared to BoNT A. However, this needs further experimental elucidation.

  6. Clostridium botulinum strains producing BoNT/F4 or BoNT/F5.

    Science.gov (United States)

    Raphael, Brian H; Bradshaw, Marite; Kalb, Suzanne R; Joseph, Lavin A; Lúquez, Carolina; Barr, John R; Johnson, Eric A; Maslanka, Susan E

    2014-05-01

    Botulinum neurotoxin type F (BoNT/F) may be produced by Clostridium botulinum alone or in combination with another toxin type such as BoNT/A or BoNT/B. Type F neurotoxin gene sequences have been further classified into seven toxin subtypes. Recently, the genome sequence of one strain of C. botulinum (Af84) was shown to contain three neurotoxin genes (bont/F4, bont/F5, and bont/A2). In this study, eight strains containing bont/F4 and seven strains containing bont/F5 were examined. Culture supernatants produced by these strains were incubated with BoNT/F-specific peptide substrates. Cleavage products of these peptides were subjected to mass spectral analysis, allowing detection of the BoNT/F subtypes present in the culture supernatants. PCR analysis demonstrated that a plasmid-specific marker (PL-6) was observed only among strains containing bont/F5. Among these strains, Southern hybridization revealed the presence of an approximately 242-kb plasmid harboring bont/F5. Genome sequencing of four of these strains revealed that the genomic backgrounds of strains harboring either bont/F4 or bont/F5 are diverse. None of the strains analyzed in this study were shown to produce BoNT/F4 and BoNT/F5 simultaneously, suggesting that strain Af84 is unusual. Finally, these data support a role for the mobility of a bont/F5-carrying plasmid among strains of diverse genomic backgrounds.

  7. New insight in the epidemiology of avian botulism outbreaks: necrophagous flies as vectors of Clostridium botulinum type C/D.

    Science.gov (United States)

    Anza, Ibone; Vidal, Dolors; Mateo, Rafael

    2014-12-01

    Avian botulism outbreaks spread through the bird carcass-maggot cycle, in which Clostridium botulinum and blowflies interact to ensure their reproduction in a mutualistic relationship where neurotoxin/spore-bearing maggot is one of the keystones. Here we investigated the hypothesis that adult blowflies may also play a significant role in botulism outbreaks by carrying C. botulinum cells between carcasses. We carried out a field experiment placing bird carcasses free of C. botulinum type C/D in containers only accessible to necrophagous flying insects in wetlands where avian botulism outbreaks were occurring and in control sites. Additionally, we performed laboratory trials to evaluate if blowflies may carry C. botulinum type C/D and for how long. Maggots bearing C. botulinum type C/D developed in 27.5% of carcasses placed in wetlands during botulism outbreaks. Calliphoridae flies in laboratory trials were able to transfer C. botulinum between two points and excreted it in their spots for up to 24 h after an infective feeding. Our results confirm that adult necrophagous flies play a role in the spreading of botulism outbreaks, which have implications in the epidemiology of this disease. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  8. An Integrative Approach to Computational Modelling of the Gene Regulatory Network Controlling Clostridium botulinum Type A1 Toxin Production.

    Science.gov (United States)

    Ihekwaba, Adaoha E C; Mura, Ivan; Walshaw, John; Peck, Michael W; Barker, Gary C

    2016-11-01

    Clostridium botulinum produces botulinum neurotoxins (BoNTs), highly potent substances responsible for botulism. Currently, mathematical models of C. botulinum growth and toxigenesis are largely aimed at risk assessment and do not include explicit genetic information beyond group level but integrate many component processes, such as signalling, membrane permeability and metabolic activity. In this paper we present a scheme for modelling neurotoxin production in C. botulinum Group I type A1, based on the integration of diverse information coming from experimental results available in the literature. Experiments show that production of BoNTs depends on the growth-phase and is under the control of positive and negative regulatory elements at the intracellular level. Toxins are released as large protein complexes and are associated with non-toxic components. Here, we systematically review and integrate those regulatory elements previously described in the literature for C. botulinum Group I type A1 into a population dynamics model, to build the very first computational model of toxin production at the molecular level. We conduct a validation of our model against several items of published experimental data for different wild type and mutant strains of C. botulinum Group I type A1. The result of this process underscores the potential of mathematical modelling at the cellular level, as a means of creating opportunities in developing new strategies that could be used to prevent botulism; and potentially contribute to improved methods for the production of toxin that is used for therapeutics.

  9. Analysis of the genetic distribution among members of Clostridium botulinum group I using a novel multilocus sequence typing (MLST) assay.

    Science.gov (United States)

    Olsen, Jaran S; Scholz, Holger; Fillo, Silvia; Ramisse, Vincent; Lista, Florigio; Trømborg, Anette K; Aarskaug, Tone; Thrane, Ingjerd; Blatny, Janet M

    2014-01-01

    Clostridium botulinum is the etiological agent of botulism. Due to food-borne poisoning and the potential use of the extremely toxic botulinum neurotoxin (BoNT) from C. botulinum in bioterror or biocrime related actions, reliable high resolution typing methods for discriminating C. botulinum strains are needed. Partial sequencing of the adk, atpH, gyrB, proC, rpoD and spo0A genes from 51 various C. botulinum/sporogenes isolates was performed, resulting in 37 different sequence types (STs). Analysis of the sequence data revealed a genetic distribution in five larger clusters with a loose correlation to the BoNT serotypes. The developed MLST assay had a slightly lower resolution ability when compared to the MLVA (multilocus variable number of tandem repeat analysis), but the two methods resulted in similar subclusters of the strains possessing the BoNT serotypes A, B and F. The current work presents the development of a novel MLST assay useful for genotyping C. botulinum related to basic phylogenetic research and trace-back analysis in microbial forensic studies. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Effect of Cultured Celery Juice, Temperature, and Product Composition on the Inhibition of Proteolytic Clostridium botulinum Toxin Production.

    Science.gov (United States)

    Golden, Max C; Wanless, Brandon J; David, Jairus R D; Kottapalli, Bala; Lineback, D Scott; Talley, Ryan J; Glass, Kathleen A

    2017-08-01

    Clostridium botulinum may be of concern in prepared refrigerated meals, for which strict cold chain management cannot be guaranteed. This study evaluated the effect of temperature, product composition, and cultured celery juice powder (CCJP) as a source of nitrite on the inhibition of botulinum toxin formation in two experimental (meat- and vegetable-based) prepared meals. Data obtained from the challenge study were compared with a published mathematical model to determine whether the model is fail-safe with regard to the tested meals. Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin at appropriate intervals in samples stored at 10, 15, or 20°C for up to 8 weeks. None of the treatments stored at 10°C for 8 weeks supported toxin production by proteolytic C. botulinum. The addition of CCJP delayed toxin production by 1 and 3 weeks in cauliflower potatoes and in Dijon pork, respectively, stored at 15°C. Toxin production was delayed by 1 week at 20°C when CCJP was added to the cauliflower potatoes. This study found that the predictive model was fail-safe but was overly conservative for the experimental meals described. Finally, this study confirms that product composition, the addition of nitrite via CCJP, storage time, and temperature play important roles in the inhibition of toxin formation by proteolytic C. botulinum.

  11. Crystallization and preliminary X-ray analysis of the HA3 component of Clostridium botulinum type C progenitor toxin

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Toshio; Tonozuka, Takashi; Kotani, Mao; Obata, Kanae [Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509 (Japan); Oguma, Keiji [Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558 (Japan); Nishikawa, Atsushi, E-mail: nishikaw@cc.tuat.ac.jp [Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509 (Japan); CREST, Japan Science and Technology Agency (Japan)

    2007-12-01

    HA3, a 70 kDa haemagglutinating protein, is a precursor form of HA3a and HA3b, the subcomponents of Clostridium botulinum type C 16S progenitor toxin. In this report, recombinant HA3 protein was overexpressed in Escherichia coli, purified and crystallized. HA3, a 70 kDa haemagglutinating protein, is a precursor form of HA3a and HA3b, the subcomponents of Clostridium botulinum type C 16S progenitor toxin. In this report, recombinant HA3 protein was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.6 Å resolution and the crystal belonged to the hexagonal space group P6{sub 3}. Matthews coefficient and self-rotation function calculations indicate that there is probably one molecule of HA3 in the asymmetric unit. A search for heavy-atom derivatives has been undertaken.

  12. Detection of Clostridium botulinum type C cells in the gastrointestinal tracts of Mozambique tilapia (Oreochromis mossambicus) by polymerase chain reaction

    Science.gov (United States)

    Nol, P.; Williamson, J.L.; Rocke, T.E.; Yuill, Thomas M.

    2004-01-01

    We established a method of directly detecting Clostridium botulinum type C cells, while minimizing spore detection, in the intestinal contents of Mozambique tilapia (Oreochromis mossambicus). This technique involved extraction of predominantly cellular DNA from tilapia intestinal tracts and used a polymerase chain reaction assay to detect presence of type C1 toxin gene. We consistently detected C. botulinum type C cells in tilapia gastrointestinal contents at a level of 7.5×104 cells per 0.25 g material or 1.9×103 cells. This technique is useful for determining prevalence of the potentially active organisms within a given population of fish and may be adapted to other types of C. botulinum and vertebrate populations as well.

  13. Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium Botulinum Strain PS-5.

    Science.gov (United States)

    Singh, Ajay K; Sachdeva, Amita; Degrasse, Jeffrey A; Croley, Timothy R; Stanker, Larry H; Hodge, David; Sharma, Shashi K

    2013-04-01

    Botulinum neurotoxins are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins. The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presence of NT genes was validated by PCR amplification of toxin specific fragments from genomic DNA of Clostridium botulinum strain PS-5 which indicated the presence of both serotype A and B genes on PS-5 genome. Further, TC was purified and characterized by Western blotting, Digoxin-enzyme linked immunosorbent assay, endopeptidase activity assay, and Liquid chromatography-Mass spectrometry. The data showed the presence of serotype A specific neurotoxin. Based on the analysis of neurotoxin genes and characterization of TC, PS-5 strain appears as a serotype A (B) strain of C. botulinum which produces only serotype A specific TC in the cell culture medium.

  14. Occurrence and distribution of Vibrio spp., Listonella spp., and Clostridium botulinum in the Seto Inland Sea of Japan.

    OpenAIRE

    Venkateswaran, K; Nakano, H; Okabe, T; Takayama, K; Matsuda, O; Hashimoto, H

    1989-01-01

    The distribution of Vibrio species in samples of surface water, bottom water (water 2 m above the sediment), and sediment from the Seto Inland Sea was studied. A simple technique using a membrane filter and short preenrichment in alkaline peptone water was developed to resuscitate the injured cells, followed by plating them onto TCBS agar. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples. Large populations of heterotrophs were found i...

  15. 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.

  16. Two-component signal transduction system CBO0787/CBO0786 represses transcription from botulinum neurotoxin promoters in Clostridium botulinum ATCC 3502.

    Science.gov (United States)

    Zhang, Zhen; Korkeala, Hannu; Dahlsten, Elias; Sahala, Elina; Heap, John T; Minton, Nigel P; Lindström, Miia

    2013-03-01

    Blocking neurotransmission, botulinum neurotoxin is the most poisonous biological substance known to mankind. Despite its infamy as the scourge of the food industry, the neurotoxin is increasingly used as a pharmaceutical to treat an expanding range of muscle disorders. Whilst neurotoxin expression by the spore-forming bacterium Clostridium botulinum appears tightly regulated, to date only positive regulatory elements, such as the alternative sigma factor BotR, have been implicated in this control. The identification of negative regulators has proven to be elusive. Here, we show that the two-component signal transduction system CBO0787/CBO0786 negatively regulates botulinum neurotoxin expression. Single insertional inactivation of cbo0787 encoding a sensor histidine kinase, or of cbo0786 encoding a response regulator, resulted in significantly elevated neurotoxin gene expression levels and increased neurotoxin production. Recombinant CBO0786 regulator was shown to bind to the conserved -10 site of the core promoters of the ha and ntnh-botA operons, which encode the toxin structural and accessory proteins. Increasing concentration of CBO0786 inhibited BotR-directed transcription from the ha and ntnh-botA promoters, demonstrating direct transcriptional repression of the ha and ntnh-botA operons by CBO0786. Thus, we propose that CBO0786 represses neurotoxin gene expression by blocking BotR-directed transcription from the neurotoxin promoters. This is the first evidence of a negative regulator controlling botulinum neurotoxin production. Understanding the neurotoxin regulatory mechanisms is a major target of the food and pharmaceutical industries alike.

  17. Environmental factors influencing the prevalence of a Clostridium botulinum type C/D mosaic strain in nonpermanent Mediterranean wetlands.

    Science.gov (United States)

    Vidal, Dolors; Anza, Ibone; Taggart, Mark A; Pérez-Ramírez, Elisa; Crespo, Elena; Hofle, Ursula; Mateo, Rafael

    2013-07-01

    Between 1978 and 2008, 13 avian botulism outbreaks were recorded in the wetlands of Mancha Húmeda (central Spain). These outbreaks caused the deaths of around 20,000 birds from over 50 species, including globally endangered white-headed ducks (Oxyura leucoceophala). Here, a significant association was found between the number of dead birds recorded in each botulism outbreak and the mean temperature in July (always >26°C). The presence of Clostridium botulinum type C/D in wetland sediments was detected by real-time PCR (quantitative PCR [qPCR]) in 5.8% of 207 samples collected between 2005 and 2008. Low concentrations of Cl(-) and high organic matter content in sediments were significantly associated with the presence of C. botulinum. Seventy-five digestive tracts of birds found dead during botulism outbreaks were analyzed; C. botulinum was present in 38.7% of them. The prevalence of C. botulinum was 18.2% (n = 22 pools) in aquatic invertebrates (Chironomidae and Corixidae families) and 33.3% (n = 18 pools) in necrophagous invertebrates (Sarcophagidae and Calliphoridae families), including two pools of adult necrophagous flies collected around bird carcasses. The presence of the bacteria in the adult fly form opens up new perspectives in the epidemiology of avian botulism, since these flies may be transporting C. botulinum from one carcass to another.

  18. Thermal and Pressure-Assisted Thermal Destruction Kinetics for Spores of Type A Clostridium botulinum and Clostridium sporogenes PA3679.

    Science.gov (United States)

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

    2016-02-01

    The purpose of this study was to determine the inactivation kinetics of the spores of the most resistant proteolytic Clostridium botulinum strains (Giorgio-A and 69-A, as determined from an earlier screening study) and of Clostridium sporogenes PA3679 and to compare the thermal and pressure-assisted thermal resistance of these spores. Spores of these strains were prepared using a biphasic medium method. C. sporogenes PA3679 spores were heat treated before spore preparation. Using laboratory-scale and pilot-scale pressure test systems, spores of Giorgio-A, 69-A, and PA3679 suspended in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer (pH 7.0) were exposed to various combinations of temperature (93 to 121°C) and pressure (0.1 to 750 MPa) to determine their resistance. More than a 5-log reduction occurred after 3 min at 113°C for spores of Giorgio-A and 69-A and after 5 min at 117°C for spores of PA3679. A combination of high temperatures (93 to 121°C) and pressures yielded greater log reductions of spores of Giorgio-A, 69-A, and PA3679 compared with reduction obtained with high temperatures alone. No survivors from initial levels (>5.0 log CFU) of Giorgio-A and 69-A were detected when processed at a combination of high temperature (117 and 121°C) and high pressure (600 and 750 MPa) for 4.5-log reduction of PA3679 spores. Thermal D-values of Giorgio-A, 69-A, and PA3679 spores decreased (i.e., 29.1 to 0.33 min for Giorgio-A, 40.5 to 0.27 min for 69-A, and 335.2 to 2.16 min for PA3679) as the temperature increased from 97 to 117°C. Pressure-assisted thermal D-values of Giorgio-A, 69-A, and PA3679 also decreased as temperature increased from 97 to 121°C at both pressures (600 and 750 MPa) (i.e., 17.19 to 0.15 min for Giorgio-A, 9.58 to 0.15 min for 69-A, and 12.93 to 0.33 min for PA3679 at 600 MPa). At higher temperatures (117 or 121°C), increasing pressure from 600 to 750 MPa had an effect on pressure-assisted thermal D-values of PA3679 (i.e., at 117

  19. Effects of gamma radiation on Clostridium botulinum type E under various parameters

    International Nuclear Information System (INIS)

    Lim, Y.H.

    1986-01-01

    Spores of Clostridium botulinum type E strain Eklund (Eklund) was irradiated with gamma radiation and its recovery was tested on the tryptone-peptone-glucose-yeast extract-agar (TPGYA) containing various levels of NaCl and Na-thioglycollate. The presence of 0.5% or more NaCl in the media decreased the viable counts, while Na-thioglycollate of up to 0.15% did not affect the recovery of both irradiated and non-irradiated spores. Eklund spores were also irradiated under air (21% O 2 ), N 2 O and N 2 , with or without the additive of one of the following agents (additive/concentration): disodium ethylenediaminetetraacetate (EDTA), 0.01 M; t-butanol, 0.1 M; NaCl, 0.01 M; catalyze, 10 mg/ml and DL-cysteine, 0.1 mM. Radiation process was most effective in destroying the spores when carried out under air (21% O 2 ), followed by N 2 O and N 2 . Among the additives tested, EDTA was the most efficient protector followed by t-butanol when irradiation process was carried under N 2 O and N 2 gas environment. Catalase and DL-cysteine sensitized the spores when irradiated under N 2 O and N 2 , while NaCl only sensitized under N 2 . Spores kept frozen at -75 0 C for 30 days but thawed prior to irradiation were more sensitive to radiation damage than freshly prepared spores. Radiation resistance of the spores increased when 15% glycerol was added to the phosphate bugger (0.06 M, pH 7.0) and used as suspending media. When the concentration of the spore increased from 10 6 /ml to 10 11 /ml, the radiosensitivities also increased. Seven strains of C. botulinum type E were screened for plasmids by agarose gel electrophoresis

  20. Food safety objective approach for controlling Clostridium botulinum growth and toxin production in commercially sterile foods.

    Science.gov (United States)

    Anderson, N M; Larkin, J W; Cole, M B; Skinner, G E; Whiting, R C; Gorris, L G M; Rodriguez, A; Buchanan, R; Stewart, C M; Hanlin, J H; Keener, L; Hall, P A

    2011-11-01

    As existing technologies are refined and novel microbial inactivation technologies are developed, there is a growing need for a metric that can be used to judge equivalent levels of hazard control stringency to ensure food safety of commercially sterile foods. A food safety objective (FSO) is an output-oriented metric that designates the maximum level of a hazard (e.g., the pathogenic microorganism or toxin) tolerated in a food at the end of the food supply chain at the moment of consumption without specifying by which measures the hazard level is controlled. Using a risk-based approach, when the total outcome of controlling initial levels (H(0)), reducing levels (ΣR), and preventing an increase in levels (ΣI) is less than or equal to the target FSO, the product is considered safe. A cross-disciplinary international consortium of specialists from industry, academia, and government was organized with the objective of developing a document to illustrate the FSO approach for controlling Clostridium botulinum toxin in commercially sterile foods. This article outlines the general principles of an FSO risk management framework for controlling C. botulinum growth and toxin production in commercially sterile foods. Topics include historical approaches to establishing commercial sterility; a perspective on the establishment of an appropriate target FSO; a discussion of control of initial levels, reduction of levels, and prevention of an increase in levels of the hazard; and deterministic and stochastic examples that illustrate the impact that various control measure combinations have on the safety of well-established commercially sterile products and the ways in which variability all levels of control can heavily influence estimates in the FSO risk management framework. This risk-based framework should encourage development of innovative technologies that result in microbial safety levels equivalent to those achieved with traditional processing methods.

  1. Regulation of neurotoxin production and sporulation by a Putative agrBD signaling system in proteolytic Clostridium botulinum.

    Science.gov (United States)

    Cooksley, Clare M; Davis, Ian J; Winzer, Klaus; Chan, Weng C; Peck, Michael W; Minton, Nigel P

    2010-07-01

    A significant number of genome sequences of Clostridium botulinum and related species have now been determined. In silico analysis of these data revealed the presence of two distinct agr loci (agr-1 and agr-2) in all group I strains, each encoding putative proteins with similarity to AgrB and AgrD of the well-studied Staphylococcus aureus agr quorum sensing system. In S. aureus, a small diffusible autoinducing peptide is generated from AgrD in a membrane-located processing event that requires AgrB. Here the characterization of both agr loci in the group I strain C. botulinum ATCC 3502 and of their homologues in a close relative, Clostridium sporogenes NCIMB 10696, is reported. In C. sporogenes NCIMB 10696, agr-1 and agr-2 appear to form transcriptional units that consist of agrB, agrD, and flanking genes of unknown function. Several of these flanking genes are conserved in Clostridium perfringens. In agreement with their proposed role in quorum sensing, both loci were maximally expressed during late-exponential-phase growth. Modulation of agrB expression in C. sporogenes was achieved using antisense RNA, whereas in C. botulinum, insertional agrD mutants were generated using ClosTron technology. In comparison to the wild-type strains, these strains exhibited drastically reduced sporulation and, for C. botulinum, also reduced production of neurotoxin, suggesting that both phenotypes are controlled by quorum sensing. Interestingly, while agr-1 appeared to control sporulation, agr-2 appeared to regulate neurotoxin formation.

  2. Prioritizing drug targets in Clostridium botulinum with a computational systems biology approach.

    Science.gov (United States)

    Muhammad, Syed Aun; Ahmed, Safia; Ali, Amjad; Huang, Hui; Wu, Xiaogang; Yang, X Frank; Naz, Anam; Chen, Jake

    2014-07-01

    A computational and in silico system level framework was developed to identify and prioritize the antibacterial drug targets in Clostridium botulinum (Clb), the causative agent of flaccid paralysis in humans that can be fatal in 5 to 10% of cases. This disease is difficult to control due to the emergence of drug-resistant pathogenic strains and the only available treatment antitoxin which can target the neurotoxin at the extracellular level and cannot reverse the paralysis. This study framework is based on comprehensive systems-scale analysis of genomic sequence homology and phylogenetic relationships among Clostridium, other infectious bacteria, host and human gut flora. First, the entire 2628-annotated genes of this bacterial genome were categorized into essential, non-essential and virulence genes. The results obtained showed that 39% of essential proteins that functionally interact with virulence proteins were identified, which could be a key to new interventions that may kill the bacteria and minimize the host damage caused by the virulence factors. Second, a comprehensive comparative COGs and blast sequence analysis of these proteins and host proteins to minimize the risks of side effects was carried out. This revealed that 47% of a set of C. botulinum proteins were evolutionary related with Homo sapiens proteins to sort out the non-human homologs. Third, orthology analysis with other infectious bacteria to assess broad-spectrum effects was executed and COGs were mostly found in Clostridia, Bacilli (Firmicutes), and in alpha and beta Proteobacteria. Fourth, a comparative phylogenetic analysis was performed with human microbiota to filter out drug targets that may also affect human gut flora. This reduced the list of candidate proteins down to 131. Finally, the role of these putative drug targets in clostridial biological pathways was studied while subcellular localization of these candidate proteins in bacterial cellular system exhibited that 68% of the

  3. Molecular gene profiling of Clostridium botulinum group III and its detection in naturally contaminated samples originating from various European countries.

    Science.gov (United States)

    Woudstra, Cedric; Le Maréchal, Caroline; Souillard, Rozenn; Bayon-Auboyer, Marie-Hélène; Anniballi, Fabrizio; Auricchio, Bruna; De Medici, Dario; Bano, Luca; Koene, Miriam; Sansonetti, Marie-Hélène; Desoutter, Denise; Hansbauer, Eva-Maria; Dorner, Martin B; Dorner, Brigitte G; Fach, Patrick

    2015-04-01

    We report the development of real-time PCR assays for genotyping Clostridium botulinum group III targeting the newly defined C. novyi sensu lato group; the nontoxic nonhemagglutinin (NTNH)-encoding gene ntnh; the botulinum neurotoxin (BoNT)-encoding genes bont/C, bont/C/D, bont/D, and bont/D/C; and the flagellin (fliC) gene. The genetic diversity of fliC among C. botulinum group III strains resulted in the definition of five major subgroups named fliC-I to fliC-V. Investigation of fliC subtypes in 560 samples, with various European origins, showed that fliC-I was predominant and found exclusively in samples contaminated by C. botulinum type C/D, fliC-II was rarely detected, no sample was recorded as fliC-III or fliC-V, and only C. botulinum type D/C samples tested positive for fliC-IV. The lack of genetic diversity of the flagellin gene of C. botulinum type C/D would support a clonal spread of type C/D strains in different geographical areas. fliC-I to fliC-III are genetically related (87% to 92% sequence identity), whereas fliC-IV from C. botulinum type D/C is more genetically distant from the other fliC types (with only 50% sequence identity). These findings suggest fliC-I to fliC-III have evolved in a common environment and support a different genetic evolution for fliC-IV. A combination of the C. novyi sensu lato, ntnh, bont, and fliC PCR assays developed in this study allowed better characterization of C. botulinum group III and showed the group to be less genetically diverse than C. botulinum groups I and II, supporting a slow genetic evolution of the strains belonging to C. botulinum group III. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  4. Evolution of Chromosomal Clostridium botulinum Type E Neurotoxin Gene Clusters: Evidence Provided by Their Rare Plasmid-Borne Counterparts.

    Science.gov (United States)

    Carter, Andrew T; Austin, John W; Weedmark, Kelly A; Peck, Michael W

    2016-03-02

    Analysis of more than 150 Clostridium botulinum Group II type E genomes identified a small fraction (6%) where neurotoxin-encoding genes were located on plasmids. Seven closely related (134-144 kb) neurotoxigenic plasmids of subtypes E1, E3, and E10 were characterized; all carried genes associated with plasmid mobility via conjugation. Each plasmid contained the same 24-kb neurotoxin cluster cassette (six neurotoxin cluster and six flanking genes) that had split a helicase gene, rather than the more common chromosomal rarA. The neurotoxin cluster cassettes had evolved as separate genetic units which had either exited their chromosomal rarA locus in a series of parallel events, inserting into the plasmid-borne helicase gene, or vice versa. A single intact version of the helicase gene was discovered on a nonneurotoxigenic form of this plasmid. The observed low frequency for the plasmid location may reflect one or more of the following: 1) Less efficient recombination mechanism for the helicase gene target, 2) lack of suitable target plasmids, and 3) loss of neurotoxigenic plasmids. Type E1 and E10 plasmids possessed a Clustered Regularly Interspaced Short Palindromic Repeats locus with spacers that recognized C. botulinum Group II plasmids, but not C. botulinum Group I plasmids, demonstrating their long-term separation. Clostridium botulinum Group II type E strains also carry nonneurotoxigenic plasmids closely related to C. botulinum Group II types B and F plasmids. Here, the absence of neurotoxin cassettes may be because recombination requires both a specific mechanism and specific target sequence, which are rarely found together. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  5. Inhibition of Clostridium botulinum in Model Reduced-Sodium Pasteurized Prepared Cheese Products.

    Science.gov (United States)

    Glass, Kathleen A; Mu, Ming; LeVine, Brian; Rossi, Frank

    2017-09-01

    The 1986 Food Research Institute-Tanaka et al. model predicts the safety of shelf-stable process cheese spread formulations using the parameters of moisture, pH, NaCl, and disodium phosphate (DSP) to inhibit toxin production by Clostridium botulinum. Although this model is very reliable for predicting safety for standard-of-identity spreads, the effects of additional factors have not been considered. The objective of this study was to create a predictive model to include the interactive effect of moisture, pH, fat, sorbic acid, and potassium-based replacements for NaCl and DSP to reflect modern reduced-sodium recipes. Eighty formulations were identified using a central composite design targeting seven factors: 50 to 60% moisture, pH 5.4 to 6.2, 0 to 0.2% sorbic acid, 10 to 30% fat, 1.7 to 2.4% NaCl, 0.8 to 1.6% DSP, and 0 to 50% potassium replacement for sodium salts. Samples were inoculated with proteolytic C. botulinum spores at 3 log spores per g, hot filled into sterile vials, and stored anaerobically at 27°C. Samples were assayed at 0, 1, 2, 3, 4, 8.5, 17.5, 26, and 40 weeks for the presence of botulinum toxin using the mouse bioassay. A parametric survival model was fit to the censored time-to-toxin data. All linear, quadratic, and pairwise effects were considered for model fit. As hypothesized, the effects of pH, sorbate, moisture, DSP, and NaCl were highly significant (P < 0.001). Fat concentration and potassium replacement effects were significant at P < 0.021 and P < 0.057, respectively. The model consistently predicted the safety failure of the toxic samples, but it also predicted failure for some samples that were not toxic. This model is an adjunct to existing models by adding the factors of potassium salts, fat, and sorbic acid to predict the botulinal safety of prepared process cheese products but is not intended to be a substitute for formulation evaluation by a competent process authority.

  6. Isolation and functional characterization of the novel Clostridium botulinum neurotoxin A8 subtype.

    Directory of Open Access Journals (Sweden)

    Skadi Kull

    Full Text Available Botulism is a severe neurological disease caused by the complex family of botulinum neurotoxins (BoNT. Based on the different serotypes known today, a classification of serotype variants termed subtypes has been proposed according to sequence diversity and immunological properties. However, the relevance of BoNT subtypes is currently not well understood. Here we describe the isolation of a novel Clostridium botulinum strain from a food-borne botulism outbreak near Chemnitz, Germany. Comparison of its botulinum neurotoxin gene sequence with published sequences identified it to be a novel subtype within the BoNT/A serotype designated BoNT/A8. The neurotoxin gene is located within an ha-orfX+ cluster and showed highest homology to BoNT/A1, A2, A5, and A6. Unexpectedly, we found an arginine insertion located in the HC domain of the heavy chain, which is unique compared to all other BoNT/A subtypes known so far. Functional characterization revealed that the binding characteristics to its main neuronal protein receptor SV2C seemed unaffected, whereas binding to membrane-incorporated gangliosides was reduced in comparison to BoNT/A1. Moreover, we found significantly lower enzymatic activity of the natural, full-length neurotoxin and the recombinant light chain of BoNT/A8 compared to BoNT/A1 in different endopeptidase assays. Both reduced ganglioside binding and enzymatic activity may contribute to the considerably lower biological activity of BoNT/A8 as measured in a mouse phrenic nerve hemidiaphragm assay. Despite its reduced activity the novel BoNT/A8 subtype caused severe botulism in a 63-year-old male. To our knowledge, this is the first description and a comprehensive characterization of a novel BoNT/A subtype which combines genetic information on the neurotoxin gene cluster with an in-depth functional analysis using different technical approaches. Our results show that subtyping of BoNT is highly relevant and that understanding of the detailed

  7. A history of study on safety of irradiated foods (2). Clostridium botulinum in irradiated seafood from the reports by the United States Atomic Energy Commission

    International Nuclear Information System (INIS)

    Miyahara, Makoto

    2004-01-01

    This review is a part of ''history of study on the wholesomeness of irradiated foods''. Clostridium botulinum in irradiated seafood have been of great concern at the beginning of development of irradiated food. This review describes the studies on Clostridium botulinum by US. Atomic Energy Commission in 1960's with their data and what they recognized it as a risk factor of irradiated foods. In 1999 FAO/IAEA/WHO reported that Clostridium botulinum type A and B spors are apparently the most resistant and thus of great concern in the radiation sterilization of food, whereas the less radiation-resistant type E spores are important in low dose irradiation of foods, particularly fishery products. This review also describes current break-through application by NASA and Canadian irradiator. (author)

  8. Uma reflexão sobre o Botulismo Alimentar (Clostridium botulinum

    Directory of Open Access Journals (Sweden)

    Marjore Patricia Ferreira Bezerra

    2016-11-01

    Full Text Available The food is of great importance to the nutritional needs of humans, becoming a vehicle for the body's metabolic activities and innocuous, making it essential to the control of food quality and hygienic conditions. It is necessary to pay attention on food safety, quality control, sanitary, hygienic conditions in which these measures must be audited by government agencies, thus avoiding the occurrence of foodborne diseases (FBD. These measures focus on prevention of these diseases, which are for biological, chemical and physical agents. The present work had as objective reporting the seriousness attached to public health through the ingestion of the toxin released by the bacterium Clostridium botulinum, emphasizing methods of preventing the development of the disease, based on the guidelines of the Sanitary and Epidemiological Surveillance. The bacteria that cause botulism, should not be associated only to eating food prepared at home, but also in restaurants and processed foods. Therefore, the botulism by contaminated food is of inestimable importance to global public health control, and preventive measures are essential to easing the incidence of cases of this disease.

  9. Mode of VAMP Substrate Recognition and Inhibition of Clostridium botulinum Neurotoxin F

    Energy Technology Data Exchange (ETDEWEB)

    Agarwal, R.; Schmidt, J; Stafford, R; Swaminathan, S

    2009-01-01

    Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exosites away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.

  10. Proteome Alterations of Hippocampal Cells Caused by Clostridium botulinum C3 Exoenzyme.

    Science.gov (United States)

    Schröder, Anke; Rohrbeck, Astrid; Just, Ingo; Pich, Andreas

    2015-11-06

    C3bot from Clostridium botulinum is a bacterial mono-ADP-ribosylating enzyme, which transfers an ADP-ribose moiety onto the small GTPases Rho A/B/C. C3bot and the catalytic inactive mutant (C3E174Q) cause axonal and dendritic growth as well as branching in primary hippocampal neurons. In cultured murine hippocampal HT22 cells, protein abundances were analyzed in response to C3bot or C3E174Q treatment using a shotgun proteomics approach. Proteome analyses were performed at four time points over 6 days. More than 4000 protein groups were identified at each time point and quantified in triplicate analyses. On day one, 46 proteins showed an altered abundance, and after 6 days, more than 700 proteins responded to C3bot with an up- or down-regulation. In contrast, C3E174Q had no provable impact on protein abundance. Protein quantification was verified for several proteins by multiple reaction monitoring. Data analysis of altered proteins revealed different cellular processes that were affected by C3bot. They are particularly involved in mitochondrial and lysosomal processes, adhesion, carbohydrate and glucose metabolism, signal transduction, and nuclear proteins of translation and ribosome biogenesis. The results of this study gain novel insights into the function of C3bot in hippocampal cells.

  11. Resistance of Spores of Clostridium botulinum 33A to Combinations of Ultraviolet and Gamma Rays1

    Science.gov (United States)

    Durban, E.; Grecz, N.

    1969-01-01

    Spores of Clostridium botulinum 33A exhibit a sigmoidal survival curve if subjected to gamma radiation. The present investigation was concerned with two questions: (i) what is the form of an ultraviolet (UV)-survival curve and (ii) what is the combined effect of UV- and gamma radiation? The UV-survival curve was found to be of sigmoidal type with a “shoulder” width of 675 ergs/mm2 and a D10 (exp) of 2,950 ergs/mm2. To test the combination effect, spores were subjected to UV doses of 225, 450, 675, and 900 ergs/mm2 followed by a series of increasing doses of gamma rays from 200 to 2,000 krad in 200-krad steps. The gamma ray-survival curves showed that increasing UV pretreatment caused a gradual loss of the “Prodiginine” yielding straight line exponential survival curves after preirradiation with UV doses of 675 ergs/mm2 and above. Simultaneously the D10 value for gamma-ray irradiation was reduced, e.g. UV preirradiation with 900 ergs/mm2 reduced the D10 by 40%. This observation emphasizes the potential practical advantage of combining UV and gamma rays for sterilization of heat-sensitive commodities. PMID:4896102

  12. Esporos de Clostridium botulinum em mel comercializado no Estado de São Paulo e em outros Estados brasileiros Clostridium botulinum spores in honey commercialized in São Paulo and other Brazilian states

    Directory of Open Access Journals (Sweden)

    Adriana Valim Ferreira Ragazani

    2008-04-01

    Full Text Available O botulismo infantil tem afetado crianças abaixo de um ano de idade em várias regiões do mundo, e o mel tem sido identificado como uma das mais importantes fontes de intoxicação alimentar. Apesar disso, há dados escassos sobre o botulismo entre crianças no Brasil, especialmente no tipo de alimento comercial mais implicado nesta patologia. Este estudo pretendeu investigar a presença de esporos de Clostridium botulinum em mel comercializado no Brasil. Cem amostras de mel comercializado em seis diferentes Estados brasileiros (SP, MG, GO, CE, MT, SC foram pesquisados para a presença de esporos de Clostridium botulinum, usando o choque térmico, seguido pela inoculação em caldo Cooked Meat Medium (Difco® e incubado em condições anaeróbias. As culturas positivas foram analisadas através de esfregaços corados pelo Gram e semeadas em placas de Reinforced Clostrideo Agar (Difco® e placas de Sulfito Polimixina Sulfadiazina -SPS (Difco®, as quais foram incubadas em condições anaeróbicas para obter colônias desta bactéria. As colônias positivas foram submetidas a teste de toxicidade através da inoculação em camundongos susceptíveis e caracterização bioquímica. Foram encontradas colônias de Clostridium botulinum que produzem toxinas ativas em 7% das amostras de mel comercial, realçando a relevância deste microrganismo para a saúde pública devido ao alto risco potencial de o mel comercializado nestas regiões brasileiras causar o botulismo infantil, especialmente em crianças abaixo de um ano de idade.Infant botulism has been affecting children under one year of age in several regions of the world and honey has been identified as one of the most important source of this food borne disease. Despite this, there are scarce data about botulism among children in Brazil, specially the type commercial food most implicated in this pathology. This study aimed at investigating the presence of spores of Clostridium botulinum in honey

  13. Mathematical modeling and Monte Carlo simulation of thermal inactivation of non-proteolytic Clostridium botulinum spores during continuous microwave-assisted pasteurization

    Science.gov (United States)

    The objective of this study is to develop a mathematical method to simulate the internal temperature history of products processed in a prototype microwave-assisted pasteurization system (MAPS) developed by Washington State University. Two products (10 oz. beef meatball trays and 16 oz. salmon fill...

  14. Construction of "Toxin Complex" in a Mutant Serotype C Strain of Clostridium botulinum Harboring a Defective Neurotoxin Gene.

    Science.gov (United States)

    Suzuki, Tomonori; Nagano, Thomas; Niwa, Koichi; Uchino, Masataka; Tomizawa, Motohiro; Sagane, Yoshimasa; Watanabe, Toshihiro

    2017-01-01

    A non-toxigenic mutant of the toxigenic serotype C Clostridium botulinum strain Stockholm (C-St), C-N71, does not produce the botulinum neurotoxin (BoNT). However, the original strain C-St produces botulinum toxin complex, in which BoNT is associated with non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin proteins (HA-70, HA-33, and HA-17). Therefore, in this study, we aimed to elucidate the effects of bont gene knockout on the formation of the "toxin complex." Nucleotide sequence analysis revealed that a premature stop codon was introduced in the bont gene, whereas other genes were not affected by this mutation. Moreover, we successfully purified the "toxin complex" produced by C-N71. The "toxin complex" was identified as a mixture of NTNHA/HA-70/HA-17/HA-33 complexes with intact NTNHA or C-terminally truncated NTNHA, without BoNT. These results indicated that knockout of the bont gene does not affect the formation of the "toxin complex." Since the botulinum toxin complex has been shown to play an important role in oral toxin transport in the human and animal body, a non-neurotoxic "toxin complex" of C-N71 may be valuable for the development of an oral drug delivery system.

  15. Two-component signal transduction system CBO0787/CBO0786 represses transcription from botulinum neurotoxin promoters in Clostridium botulinum ATCC 3502.

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    2013-03-01

    Full Text Available Blocking neurotransmission, botulinum neurotoxin is the most poisonous biological substance known to mankind. Despite its infamy as the scourge of the food industry, the neurotoxin is increasingly used as a pharmaceutical to treat an expanding range of muscle disorders. Whilst neurotoxin expression by the spore-forming bacterium Clostridium botulinum appears tightly regulated, to date only positive regulatory elements, such as the alternative sigma factor BotR, have been implicated in this control. The identification of negative regulators has proven to be elusive. Here, we show that the two-component signal transduction system CBO0787/CBO0786 negatively regulates botulinum neurotoxin expression. Single insertional inactivation of cbo0787 encoding a sensor histidine kinase, or of cbo0786 encoding a response regulator, resulted in significantly elevated neurotoxin gene expression levels and increased neurotoxin production. Recombinant CBO0786 regulator was shown to bind to the conserved -10 site of the core promoters of the ha and ntnh-botA operons, which encode the toxin structural and accessory proteins. Increasing concentration of CBO0786 inhibited BotR-directed transcription from the ha and ntnh-botA promoters, demonstrating direct transcriptional repression of the ha and ntnh-botA operons by CBO0786. Thus, we propose that CBO0786 represses neurotoxin gene expression by blocking BotR-directed transcription from the neurotoxin promoters. This is the first evidence of a negative regulator controlling botulinum neurotoxin production. Understanding the neurotoxin regulatory mechanisms is a major target of the food and pharmaceutical industries alike.

  16. Effect of headspace CO2 concentration on toxin production by Clostridium botulinum in MAP, irradiated fresh pork

    International Nuclear Information System (INIS)

    Lambert, A.D.; Smith, J.P.; Dodds, K.L.

    1991-01-01

    The effects of five initial levels of CO2 (15, 30, 45, 60, and 75%) and three irradiation doses (0, 0.5, and 1.0 kGy) on toxin production by Clostridium botulinum in inoculated fresh pork were studied using factorial design experiments. Headspace CO2 levels increased in all samples during storage at 15 degrees C. In most treatments, spoilage preceded toxigenesis. Toxin production occurred faster in samples initially packaged with 15 to 30% of CO2 while higher levels of CO2 (45-75%) delayed toxin production. Low-dose irradiation delayed toxin production at all levels of CO2 in the package headspace. Contrary to expectations, including a CO2 absorbent in the package enhanced toxin production by C. botulinum. This was attributed to production of H2 by the CO2 absorbent, possibly resulting in a decrease in the oxido-reduction potential of the meat

  17. Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.

    Science.gov (United States)

    Kalb, Suzanne R; Baudys, Jakub; Smith, Theresa J; Smith, Leonard A; Barr, John R

    2014-04-01

    Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.

  18. Produção e caracterização de quimeras recombinantes C e D de Clostridium botulinum

    OpenAIRE

    Gil, Luciana Aquini Fernandes

    2012-01-01

    O botulismo bovino é uma intoxicação letal causada pela ingestão da neurotoxina produzida pelo Clostridium botulinum principalmente dos tipos C e D que atua inibindo a liberação de acetilcolina na junção neuromuscular levando à morte por paralisia flácida, com grande importância econômica e sanitária, sendo uma das principais causas de morte em bovinos adultos no Brasil. O controle imunológico do botulismo bovino depende da presença de anticorpos neutralizantes contra as neurotoxinas botul...

  19. The role of small acid-soluble proteins (SASPs) in protection of spores of Clostridium botulinum against nitrous acid.

    Science.gov (United States)

    Meaney, Carolyn A; Cartman, Stephen T; McClure, Peter J; Minton, Nigel P

    2016-01-04

    Mutant strains of Clostridium botulinum ATCC 3502 were generated using the ClosTron in four genes (CBO1789, CBO1790, CBO3048, CBO3145) identified as encoding α/β-type SASP homologues. The spores of mutant strains in which CBO1789 or CBO1790 was inactivated demonstrated a significant increase in sensitivity to the damaging agent nitrous acid (P0.05), two other chemicals commonly used as components of disinfection regimes. These data indicate that the SASPs CBO1789 or CBO1790 play a significant role in resistance to nitrous acid, but not in resistance to formaldehyde or hydrogen peroxide. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Identification and characterization of Clostridium botulinum group III field strains by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS).

    Science.gov (United States)

    Bano, Luca; Drigo, Ilenia; Tonon, Elena; Pascoletti, Simone; Puiatti, Cinzia; Anniballi, Fabrizio; Auricchio, Bruna; Lista, Florigio; Montecucco, Cesare; Agnoletti, Fabrizio

    2017-12-01

    Animal botulism is primarily due to botulinum neurotoxin (BoNT) types C, D or their chimeric variants C/D or D/C, produced by Clostridium botulinum group III, which appears to include the genetically indistinguishable Clostridium haemolyticum and Clostridium novyi. In the present study, we used matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI TOF MS) to identify and characterize 81 BoNT-producing Clostridia isolated in 47 episodes of animal botulism. The instrument's default database, containing no entries for Clostridium botulinum, permitted reliable identification of 26 strains at the genus level. Although supplementation of the database with reference strains enhanced the instrument's ability to identify the neurotoxic strains at the genus level, resolution was not sufficient to recognize field strains at species level. Characterization by MALDI TOF confirmed the well-documented phenotypic and genetic differences between Clostridium botulinum strains of serotypes normally implicated in human botulism (A, B, E, F) and other Clostridium species able to produce BoNTs type C and D. The chimeric and non-chimeric field strains grouped separately. In particular, very low similarity was found between two non-chimeric type C field strains isolated in the same outbreak and the other field strains. This difference was comparable with the differences among the various Clostridia species included in the study. Characterization by MALDI TOF confirmed that BoNT-producing Clostridia isolated from animals are closely related and indistinguishable at the species level from Clostridium haemolyticum and Clostridium novyi reference strains. On the contrary, there seem to be substantial differences among chimeric and some non-chimeric type C strains. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Medical Countermeasure Models. Volume 8. Botulinum Neurotoxin

    Science.gov (United States)

    2013-04-12

    neurotoxin is produced primarily by the bacterial species Clostridium botulinum , although Clostridium baratii and Clostridium butyricum are also capable of...varied from study to study and ranged from 0.02 IU/ml to 0.2 IU/ml. 54 Fiock MA et al., “Studies of Immunity to Toxins of Clostridium Botulinum . IX... Clostridium Botulinum . IX. Immunologic Response of Man to Purified Pentavalent ABCDE Botulinum Toxoid.” The Journal of Immunology. 90(5). 1962. 66 Siegel

  2. Synergistic capture of Clostridium botulinum Type A neurotoxin by scFv antibodies to novel epitopes

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Sean A.; Barr, John R.; Kalb, Suzanne R.; Marks, James D.; Baird, Cheryl L.; Cangelosi, Gerard A.; Miller, Keith D.; Feldhaus, Michael J.

    2011-10-01

    A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, three also bound to full-length BoNT/A toxin complex with affinities ranging from 5 nM to 170 nM. Epitope binning showed that the three unique clones recognized at least two epitopes that were distinct from one another and from the detection MAbs. After production in E. coli, the scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigen. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support.

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

    Science.gov (United States)

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

    2015-06-01

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

  4. Phenotypic characterization of Clostridium botulinum strains isolated from infant botulism cases in Argentina Caracterización fenotípica de cepas de Clostridium botulinum aisladas de casos de botulismo del lactante en Argentina

    Directory of Open Access Journals (Sweden)

    M. D. Sagua

    2009-09-01

    Full Text Available Infant botulism is the most common form of human botulism; however, its transmission has not been completely explained yet. Some of the most recognized potential sources of Clostridium botulinum spores are the soil, dust, honey and medicinal herbs. In Argentina, 456 cases of infant botulism were reported between 1982 and 2007. C. botulinum type A was identified in 455 of these cases whereas type B was identified in just one case. However, in Argentina, types A, B, E, F, G, and Af have been isolated from environmental sources. It is not clearly known if strains isolated from infant botulism cases have different characteristics from strains isolated from other sources. During this study, 46 C. botulinum strains isolated from infant botulism cases and from environmental sources were typified according to phenotypic characteristics. Biochemical tests, antimicrobial activity, and haemagglutinin-negative botulinum neurotoxin production showed uniformity among all these strains. Despite the variability observed in the botulinum neurotoxin's binding to cellular receptors, no correlation was found between these patterns and the source of the botulinum neurotoxin. However, an apparent geographical clustering was observed, since strains isolated from Argentina had similar characteristics to those isolated from Italy and Japan, but different to those isolated from the United States.El botulismo del lactante es la forma más común del botulismo humano; sin embargo, su forma de transmisión no ha sido totalmente explicada. El suelo, el polvo ambiental, la miel y algunas hierbas medicinales son potenciales fuentes de esporas de Clostridium botulinum. Entre 1982 y 2007 se informaron en Argentina 456 casos de botulismo del lactante, 455 casos debidos al serotipo A y uno al serotipo B. Sin embargo, los serotipos A, B, E, F, G y Af han sido aislados de suelos y otras fuentes en Argentina. No se conoce si las cepas aisladas de casos de botulismo del lactante

  5. Investigations into an Outbreak of Botulism Caused by Clostridium botulinum Type C/D in Laying Hens.

    Science.gov (United States)

    Skarin, Hanna; Lindgren, Ylva; Jansson, Désirée S

    2015-06-01

    This case report describes a recent botulism outbreak in commercial laying hens with a history of increased mortality and flaccid paralysis. Routine diagnostic gross examination and microscopy from seven hens were inconclusive, but botulinum neurotoxin (BoNT) in peripheral blood was neutralized with both type C and type D antitoxins in the mouse bioassay. During a farm visit, 10 additional hens from a 34-wk-old flock on the farm were selected for clinical examination and further sampling. Nine hens were observed in sternal recumbency, with flaccid paralysis of the neck, drooping wings and tail, inability to escape, and bilateral ptosis, and one hen showed nonspecific clinical signs. Samples from cecum and liver were collected, and the gene coding for BoNT was detected by PCR in all 10 cecal samples and in four of the liver samples. Clostridium botulinum mosaic type C/D was isolated from 5 out of 10 hens from either cecum or liver, and the isolates were subjected to pulsed-field gel electrophoresis subtyping. All five isolates produced the same banding pattern, which was identical or showed >90% similarity to isolates from three different outbreaks on broiler farms in Sweden and Denmark during the 2007-10 period. However, they were clearly distinguishable from the predominantly reported pulsotype associated with avian botulism outbreaks in Europe. The authors are unaware of any previous report of C. botulinum mosaic type C/D isolates from laying hens.

  6. The two-component system CBO2306/CBO2307 is important for cold adaptation of Clostridium botulinum ATCC 3502.

    Science.gov (United States)

    Derman, Yağmur; Isokallio, Marita; Lindström, Miia; Korkeala, Hannu

    2013-10-01

    Clostridium botulinum is a notorious foodborne pathogen. Its ability to adapt to and grow at low temperatures is of interest for food safety. Two-component systems (TCSs) have been reported to be involved in cold-shock and growth at low temperatures. Here we show the importance of TCS CBO2306/CBO2307 in the cold-shock response of C. botulinum ATCC 3502. The relative expression levels of the cbo2306 and cbo2307 were up to 4.4-fold induced in the cold-shocked cultures but negatively regulated in the late-log and stationary growth phase in relation to early logarithmic growth phase in non-shocked cultures. Importance of the CBO2306/CBO2307 in the cold stress was further demonstrated by impaired growth of insertional cbo2306 or cbo2307 knockout mutants in relation to the wild-type strain ATCC 3502. The results suggest that the TCS CBO2306/CBO2307 is important for cold-shock response and adaptation of C. botulinum ATCC 3502 to low temperature. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Structural analysis of Clostridium botulinum neurotoxin type D as a platform for the development of targeted secretion inhibitors.

    Science.gov (United States)

    Masuyer, Geoffrey; Davies, Jonathan R; Moore, Kevin; Chaddock, John A; Ravi Acharya, K

    2015-09-01

    The botulinum neurotoxin type D is one of seven highly potent toxins produced by Clostridium botulinum which inhibit neurotransmission at cholinergic nerve terminals. A functional fragment derived from the toxin, LHn, consisting of the catalytic and translocation domains, has been heralded as a platform for the development of targeted secretion inhibitors. These secretion inhibitors are aimed at retargeting the toxin towards a specific cell type to inhibit vesicular secretion. Here we report crystal structures of LHn from serotype D at 2.3 Å, and that of SXN101959 at 3.1 Å resolution. SXN101959, a derivative that combines LHn from serotype D with a fragment of the growth hormone releasing hormone, has previously revealed promising results in inhibiting growth hormone release in pituitary somatotrophs. These structures offer for the first time insights into the translocation domain interaction with the catalytic domain in serotype D. Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B, and D. Taken together, these results demonstrate the robustness of the 'LHn fold' across serotypes and its use in engineering additional polypeptide components with added functionality. Our study demonstrates the suitability of botulinum neurotoxin, and serotype D in particular, as a basis for engineering novel secretion inhibitors.

  8. Toxin occurrence time in relation to sensorial changes in meat cans contaminated with Clostridium botulinum type B endospores.

    Science.gov (United States)

    Palec, W

    1996-01-01

    The study concerning dissemination and expansion of spore-forming rods and the possibility of botulinum toxin occurrence was performed on meat cans model contaminated experimentally with Clostridium botulinum type B366 toxigenic strain endospores. Meat cans were contaminated with the spore suspension of 4 x 10(3) spores per can and they were stored at 30 degrees C. Methodically visible toxigenicity (biological assay performed on white mice) took place in 84.3% cases by the time deformation of contaminated cans took place. When the first can bulging occurred almost 50% of contaminated cans were sensorially estimated as useful for consumption but almost 40% of them already contained toxin. In comparison to contaminated cans estimated sensorially as useful for consumption (in each experimental group) the percentage was established where the presence of toxin was determined as "consumer's risk". The mean value of those percentages was 76.4% for all experimental groups. In the studies on the vegetation dynamics and C. botulinum rods and toxin expansion in contaminated cans, we demonstrated the spore-forming rods dissemination, as well as limited range of toxin occurrence. Undertaken studies demonstrate that the process of toxigenicity often precede of accumulation of metabolic gases responsible for cans' deformation, as well as generation of visible sensorial changes which disqualified the contaminated cans for consumption.

  9. Clostridium botulinum neurotoxin type B is heat-stable in milk and not inactivated by pasteurization

    Science.gov (United States)

    Foodborne botulism is caused by the ingestion of foods containing botulinum neurotoxins (BoNTs). Currently, the only accepted assay to detect active C. botulinum neurotoxin is an in vivo mouse bioassay, which raises ethical concerns with regard to the use of experimental animals. Therefore, there is...

  10. Combined high pressure and thermal processing on inactivation of type A and proteolytic type B spores of Clostridium botulinum.

    Science.gov (United States)

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

    2013-08-01

    The aim of this study was to determine the resistance of multiple strains of Clostridium botulinum type A and proteolytic type B spores exposed to combined high pressure and thermal processing and compare their resistance with Clostridium sporogenes PA3679 and Bacillus amyloliquefaciens TMW-2.479-Fad-82 spores. The resistance of spores suspended in N-(2acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7.0) was determined at a process temperature of 105°C, with high pressures of 600, 700, and 750 MPa by using a laboratory-scale pressure test system. No surviving spores of the proteolytic B strains were detected after processing at 105°C and 700 MPa for 6 min. A . 7-log reduction of B. amyloliquefaciens spores was observed when processed for 4 min at 105°C and 700 MPa. D-values at 105°C and 700 MPa for type A strains ranged from 0.57 to 2.28 min. C. sporogenes PA3679 had a D-value of 1.48 min at 105°C and 700 MPa. Spores of the six type A strains with high D-values along with C. sporogenes PA3679 and B. amyloliquefaciens were further evaluated for their pressure resistance at pressures 600 and 750 MPa at 105°C. As the process pressure increased from 600 to 750 MPa at 105°C, D-values of some C. botulinum strains and C. sporogenes PA3679 spores decreased (i.e., 69-A, 1.91 to 1.33 min and PA3679, 2.35 to 1.29 min). Some C. botulinum type A strains were more resistant than C. sporogenes PA3679 and B. amyloliquefaciens to combined high pressure and heat, based on D-values determined at 105°C. Pulsed-field gel electrophoresis (PFGE) was also performed to establish whether strains with a similar restriction banding pattern also exhibited similar D-values. However, no correlation between the genomic background of a strain and its resistance to high pressure processing was observed, based on PFGE analysis. Spores of proteolytic type B strains of C. botulinum were less resistant to combined high pressure and heat (700 MPa and 105°C) treatment when

  11. Structural Studies on Intact Clostridium Botulinum Neurotoxins Complexed with Inhibitors Leading to Drug Design

    National Research Council Canada - National Science Library

    Swaminathan, Subramanyam

    2006-01-01

    .... Accordingly it must be possible to design a common drug for all of them. We have determined the structure of the C fragment of botulinum neurotoxin type B and found that the N-terminal helix reorients...

  12. Structural Studies on Intact Clostridium Botulinum Neurotoxins Complexed with Inhibitors Leading to Drug Design

    National Research Council Canada - National Science Library

    Swaminathan, Subramanyam

    2005-01-01

    .... While one is common to botulinum toxins, the other is unique for tetanus. The second unique site also binds a tri-peptide which suggests that this peptide could be used as an inhibitor for tetanus, at least...

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

    OpenAIRE

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

    2014-01-01

    Clostridium botulinum produces heat-resistant endospores that may germinate and outgrow into neurotoxic cultures in foods. Sporulation is regulated by the transcription factor Spo0A and the alternative sigma factors SigF, SigE, SigG, and SigK in most spore formers studied to date. We constructed mutants of sigF, sigE, and sigG in C. botulinum ATCC 3502 and used quantitative reverse transcriptase PCR and electron microscopy to assess their expression of the sporulation pathway on transcription...

  14. BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani.

    Science.gov (United States)

    Raffestin, Stéphanie; Dupuy, Bruno; Marvaud, Jean Christophe; Popoff, Michel R

    2005-01-01

    Clostridium botulinum and Clostridium tetani, respectively, produce potent toxins, botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeTx), which are responsible for severe diseases, botulism and tetanus. Neurotoxin synthesis is a regulated process in Clostridium. The genes botR/A in C. botulinum A and tetR in C. tetani positively regulate expression of BoNT/A and associated non-toxic proteins (ANTPs), as well as TeTx respectively. The botR/A gene lies in close vicinity of the two operons which contain bont/A and antps genes in C. botulinum A, and tetR immediately precedes the tetX gene in C. tetani. We show that BotR/A and TetR function as specific alternative sigma factors rather than positive regulators based on the following results: (i) BotR/A and TetR associated with target DNAs only in the presence of the RNA polymerase core enzyme (Core), (ii) BotR/A and TetR directly bound with the core enzyme, (iii) BotR/A-Core recognized -35 and -10 regions of ntnh-bont/A promoter and (iv) BotR/A and TetR triggered in vitro transcription from the target promoters. In C. botulinum A, bont/A and antps genes are transcribed as bi- and tricistronic operons controlled by BotR/A. BotR/A and TetR are seemingly related to a new subgroup of the sigma70 family that includes TcdR and UviA, which, respectively, regulate production of toxins A and B in C. difficile and bacteriocin in C. perfringens. Sequences of -35 region are highly conserved in the promoter of target toxin genes in C. botulinum, C. tetani, C. difficile and C. perfringens. Overall, a common regulation mechanism probably controls toxin gene expression in these four toxigenic clostridial species.

  15. Prevalence of neurotoxic Clostridium botulinum type C in the gastrointestinal tracts of tilapis (Oreochromis mossambicus) in the Salton Sea

    Science.gov (United States)

    Nol, P.J.; Rocke, T.E.; Gross, K.; Yuill, Thomas M.

    2004-01-01

    Tilapia (Oreochromis mossambicus) have been implicated as the source of type C toxin in avian botulism outbreaks in pelicans (Pelecanus erythrorhynchos, Pelecanus occidentalis californicus) at the Salton Sea in southern California (USA). We collected sick, dead, and healthy fish from various sites throughout the Sea during the summers of 1999 through 2001 and tested them for the presence of Clostridium botulinum type C cells by polymerase chain reaction targeting the C1 neurotoxin gene. Four of 96 (4%), 57 of 664 (9%), and five of 355 (1%) tilapia tested were positive for C. botulinum type C toxin gene in 1999, 2000, and 2001, respectively. The total number of positive fish was significantly greater in 2000 than in 2001 (P<0.0001). No difference in numbers of positives was detected between sick and dead fish compared with live fish. In 2000, no significant relationships were revealed among the variables studied, such as location and date of collection.

  16. Expression and purification of neurotoxin-associated protein HA-33/A from Clostridium botulinum and evaluation of its antigenicity.

    Science.gov (United States)

    Sayadmanesh, Ali; Ebrahimi, Firouz; Hajizade, Abbas; Rostamian, Mosayeb; Keshavarz, Hani

    2013-01-01

    Botulinum neurotoxin (BoNT) complexes consist of neurotoxin and neurotoxin-associated proteins. Hemagglutinin-33 (HA-33) is a member of BoNT type A (BoNT/A) complex. Considering the protective role of HA-33 in preservation of BoNT/A in gastrointestinal harsh conditions and also its adjuvant role, recombinant production of this protein is favorable. Thus in this study, HA-33 was expressed and purified, and subsequently its antigenicity in mice was studied. Initially, ha-33 gene sequence of Clostridium botulinum serotype A was adopted from GenBank. The gene sequence was optimized and synthesized in pET28a (+) vector. E. coli BL21 (DE3) strain was transformed by the recombinant vector and the expression of HA-33 was optimized at 37°C and 5 h induction time. The recombinant protein was purified by nickel nitrilotriacetic acid agarose affinity chromatography and confirmed by immunoblotting. Enzyme Linked Immunoassay showed a high titer antibody production in mice. The results indicated a highly expressed and purified recombinant protein, which is able to evoke high antibody titers in mice.

  17. Heat shock and prolonged heat stress attenuate neurotoxin and sporulation gene expression in group I Clostridium botulinum strain ATCC 3502.

    Science.gov (United States)

    Selby, Katja; Mascher, Gerald; Somervuo, Panu; Lindström, Miia; Korkeala, Hannu

    2017-01-01

    Foodborne pathogenic bacteria are exposed to a number of environmental stresses during food processing, storage, and preparation, and in the human body. In order to improve the safety of food, the understanding of molecular stress response mechanisms foodborne pathogens employ is essential. Many response mechanisms that are activated during heat shock may cross-protect bacteria against other environmental stresses. To better understand the molecular mechanisms Clostridium botulinum, the causative agent of botulism, utilizes during acute heat stress and during adaptation to stressfully high temperature, the C. botulinum Group I strain ATCC 3502 was grown in continuous culture at 39°C and exposed to heat shock at 45°C, followed by prolonged heat stress at 45°C to allow adaptation of the culture to the high temperature. Growth in continuous culture was performed to exclude secondary growth phase effects or other environmental impacts on bacterial gene transcription. Changes in global gene expression profiles were studied using DNA microarray hybridization. During acute heat stress, Class I and III heat shock genes as well as members of the SOS regulon were activated. The neurotoxin gene botA and genes encoding the neurotoxin-associated proteins were suppressed throughout the study. Prolonged heat stress led to suppression of the sporulation machinery whereas genes related to chemotaxis and motility were activated. Induced expression of a large proportion of prophage genes was detected, suggesting an important role of acquired genes in the stress resistance of C. botulinum. Finally, changes in the expression of a large number of genes related to carbohydrate and amino acid metabolism indicated remodeling of the cellular metabolism.

  18. Structure, Function and Evolution of Clostridium botulinum C2 and C3 Toxins: Insight to Poultry and Veterinary Vaccines.

    Science.gov (United States)

    Chellapandi, Paulchamy; Prisilla, Arokiyasamy

    2017-01-01

    Clostridium botulinum group III strains are able to produce cytotoxins, C2 toxin and C3 exotoxin, along with botulinum neurotoxin types C and D. C2 toxin and C3 exotoxin produced by this organism are the most important members of bacterial ADP-ribosyltransferase superfamily. Both toxins have distinct pathophysiological functions in the avian and mammalian hosts. The members of this superfamily transfer an ADP-ribose moiety of NAD+ to specific eukaryotic target proteins. The present review describes the structure, function and evolution aspects of these toxins with a special emphasis to the development of veterinary vaccines. C2 toxin is a binary toxin that consists of a catalytic subunit (C2I) and a translocation subunit (C2II). C2I component is structurally and functionally similar to the VIP2 and iota A toxin whereas C2II component shows a significant homology with the protective antigen from anthrax toxin and iota B. Unlike C2 toxin, C3 toxin is devoid of translocation/binding subunit. Extensive studies on their sequence-structure-function link spawn additional efforts to understand the catalytic mechanisms and target recognition. Structural and functional relationships with them are often determined by using evolutionary constraints as valuable biological measures. Enzyme-deficient mutants derived from these toxins have been used as drug/protein delivery systems in eukaryotic cells. Thus, current knowledge on their molecular diversity is a well-known perspective to design immunotoxin or subunit vaccine for C. botulinum infection. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. Genomic Epidemiology of Clostridium botulinum Isolates from Temporally Related Cases of Infant Botulism in New South Wales, Australia.

    Science.gov (United States)

    McCallum, Nadine; Gray, Timothy J; Wang, Qinning; Ng, Jimmy; Hicks, Leanne; Nguyen, Trang; Yuen, Marion; Hill-Cawthorne, Grant A; Sintchenko, Vitali

    2015-09-01

    Infant botulism is a potentially life-threatening paralytic disease that can be associated with prolonged morbidity if not rapidly diagnosed and treated. Four infants were diagnosed and treated for infant botulism in NSW, Australia, between May 2011 and August 2013. Despite the temporal relationship between the cases, there was no close geographical clustering or other epidemiological links. Clostridium botulinum isolates, three of which produced botulism neurotoxin serotype A (BoNT/A) and one BoNT serotype B (BoNT/B), were characterized using whole-genome sequencing (WGS). In silico multilocus sequence typing (MLST) found that two of the BoNT/A-producing isolates shared an identical novel sequence type, ST84. The other two isolates were single-locus variants of this sequence type (ST85 and ST86). All BoNT/A-producing isolates contained the same chromosomally integrated BoNT/A2 neurotoxin gene cluster. The BoNT/B-producing isolate carried a single plasmid-borne bont/B gene cluster, encoding BoNT subtype B6. Single nucleotide polymorphism (SNP)-based typing results corresponded well with MLST; however, the extra resolution provided by the whole-genome SNP comparisons showed that the isolates differed from each other by >3,500 SNPs. WGS analyses indicated that the four infant botulism cases were caused by genomically distinct strains of C. botulinum that were unlikely to have originated from a common environmental source. The isolates did, however, cluster together, compared with international isolates, suggesting that C. botulinum from environmental reservoirs throughout NSW have descended from a common ancestor. Analyses showed that the high resolution of WGS provided important phylogenetic information that would not be captured by standard seven-loci MLST. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. Science, practice, and human errors in controlling Clostridium botulinum in heat-preserved food in hermetic containers.

    Science.gov (United States)

    Pflug, Irving J

    2010-05-01

    The incidence of botulism in canned food in the last century is reviewed along with the background science; a few conclusions are reached based on analysis of published data. There are two primary aspects to botulism control: the design of an adequate process and the delivery of the adequate process to containers of food. The probability that the designed process will not be adequate to control Clostridium botulinum is very small, probably less than 1.0 x 10(-6), based on containers of food, whereas the failure of the operator of the processing equipment to deliver the specified process to containers of food may be of the order of 1 in 40, to 1 in 100, based on processing units (retort loads). In the commercial food canning industry, failure to deliver the process will probably be of the order of 1.0 x 10(-4) to 1.0 x 10(-6) when U.S. Food and Drug Administration (FDA) regulations are followed. Botulism incidents have occurred in food canning plants that have not followed the FDA regulations. It is possible but very rare to have botulism result from postprocessing contamination. It may thus be concluded that botulism incidents in canned food are primarily the result of human failure in the delivery of the designed or specified process to containers of food that, in turn, result in the survival, outgrowth, and toxin production of C. botulinum spores. Therefore, efforts in C. botulinum control should be concentrated on reducing human errors in the delivery of the specified process to containers of food.

  1. Insights into the evolutionary origins of clostridial neurotoxins from analysis of the Clostridium botulinum strain A neurotoxin gene cluster.

    Science.gov (United States)

    Doxey, Andrew C; Lynch, Michael D J; Müller, Kirsten M; Meiering, Elizabeth M; McConkey, Brendan J

    2008-11-14

    Clostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Despite considerable progress in understanding CNT structure and function, the evolutionary origins of CNTs remain a mystery as they are unique to Clostridium and possess a sequence and structural architecture distinct from other protein families. Uncovering the origins of CNTs would be a significant contribution to our understanding of how pathogens evolve and generate novel toxin families. The C. botulinum strain A genome was examined for potential homologues of CNTs. A key link was identified between the neurotoxin and the flagellin gene (CBO0798) located immediately upstream of the BoNT/A neurotoxin gene cluster. This flagellin sequence displayed the strongest sequence similarity to the neurotoxin and NTNH homologue out of all proteins encoded within C. botulinum strain A. The CBO0798 gene contains a unique hypervariable region, which in closely related flagellins encodes a collagenase-like domain. Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity. Additional links to collagenase-related sequences and functions were detected by further analysis of CNTs and surrounding genes, including sequence similarities to collagen-adhesion domains and collagenases. Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains. Multiple lines of evidence suggest that the neurotoxin and adjacent genes evolved from an ancestral collagenase-like gene cluster, linking CNTs to another major family of clostridial proteolytic toxins. Duplication, reshuffling and assembly of neighboring genes within the BoNT/A neurotoxin gene cluster may have lead to the neurotoxin's unique architecture. This work provides new insights into the evolution of C

  2. Clostridium sporogenes PA 3679 and its uses in the derivation of thermal processing schedules for low-acid shelf-stable foods and as a research model for proteolytic Clostridium botulinum.

    Science.gov (United States)

    Brown, Janelle L; Tran-Dinh, Nai; Chapman, Belinda

    2012-04-01

    The putrefactive anaerobe Clostridium sporogenes PA 3679 has been widely used as a nontoxigenic surrogate for proteolytic Clostridium botulinum in the validation of thermal processes for low-acid shelf-stable foods, as a target organism in the derivation of thermal processes that reduce the risk of spoilage of such foods to an acceptable level, and as a research model for proteolytic strains of C. botulinum. Despite the importance of this organism, our knowledge of it has remained fragmented. In this article we draw together the literature associated with PA 3679 and discuss the identity of this organism, the phylogenetic relationships that exist between PA 3679 and various strains of C. sporogenes and proteolytic C. botulinum, the heat resistance characteristics of PA 3679, the advantages and limitations associated with its use in the derivation of thermal processing schedules, and the knowledge gaps and opportunities that exist with regard to its use as a research model for proteolytic C. botulinum. Phylogenetic analysis reviewed here suggests that PA 3679 is more closely related to various strains of proteolytic C. botulinum than to selected strains, including the type strain, of C. sporogenes. Even though PA 3679 is demonstrably nontoxigenic, the genetic basis of this nontoxigenic status remains to be elucidated, and the genetic sequence of this microorganism appears to be the key knowledge gap remaining to be filled. Our comprehensive review of comparative heat resistance data gathered for PA 3679 and proteolytic strains of C. botulinum over the past 100 years supports the practice of using PA 3679 as a (typically fail-safe) thermal processing surrogate for proteolytic C. botulinum.

  3. Role of csp genes in NaCl, pH, and ethanol stress response and motility in Clostridium botulinum ATCC 3502.

    Science.gov (United States)

    Derman, Yağmur; Söderholm, Henna; Lindström, Miia; Korkeala, Hannu

    2015-04-01

    Clostridium botulinum is a notable food pathogen and responsible for botulism due to production of botulinum neurotoxin. Strains of C. botulinum can adapt to and survive in stress conditions and food processing. The cold shock protein coding genes (csp) are involved in growth at low temperature, but they may also possess other functions. In this mutational analysis we show that cspB and cspC, but not cspA, are important for NaCl, pH and ethanol stress responses and for motility of C. botulinum ATCC 3502. In all NaCl concentrations tested, the cspB mutant had lower maximum growth rate and, together with the cspC mutant, a longer lag phase compared to the wild-type strain. At low pH, the cspB and cspC mutants showed either lower maximum growth rates or longer lag phases compared to the wild type. In all ethanol concentrations tested, the cspB mutant had lower maximum growth rates and the cspC mutant had a longer lag phase than the wild-type strain. Motility was reduced in cspA and cspC mutants, and flagella formation was affected. The results suggest that cspB plays a universal role in stress response and cspC aids C. botulinum in NaCl, pH and ethanol stress in C. botulinum ATCC 3502. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Contamination of honey produced in the Republic of Kazakhstan with Clostridium botulinum

    Directory of Open Access Journals (Sweden)

    Mustafina Raikhan

    2015-06-01

    Full Text Available The paper presents the first results of a study on the contamination of honey produced in the Republic of Kazakhstan with C. botulinum spores known to pose a potential infection threat to infants. During microbiological analysis, culturing methods with TPGY, Willis-Hobbs agar, FAA agar connected with PCR, sequencing, and a mouse bioassay were used. The C. botulinum contamination rate of honey was relatively low as determined, at 0.91%. Nonetheless, the potential danger of the bacteria to childrens’ health should not be neglected

  5. Quantitative real-time PCR for detection of neurotoxin genes of Clostridium botulinum types A, B and C in equine samples.

    Science.gov (United States)

    Johnson, Amy L; McAdams-Gallagher, Susan C; Sweeney, Raymond W

    2014-01-01

    Botulism in horses in the USA is attributed to Clostridium botulinum types A, B or C. In this study, a duplex quantitative real-time PCR (qPCR) for detection of the neurotoxin genes of C. botulinum types A and B, and a singleplex qPCR for detection of the neurotoxin gene of C. botulinum type C, were optimized and validated for equine gastrointestinal, faecal and feed samples. The performance of these assays was evaluated and compared to the standard mouse bioassay (MBA) using 148 well-characterized samples, most of which were acquired from a repository of veterinary diagnostic samples from cases of botulism: 106 samples positive for C. botulinum (25 type A, 27 type B, 28 type C, 1 type D and 25 type E) and 42 negative samples. The sensitivities of the qPCR assays were 89%, 86% and 96% for C. botulinum types A, B and C, respectively. The overall sensitivity of the mouse bioassay for types A, B and C was 81%. The specificities of the qPCR assays were 99-100% and the specificity of the mouse bioassay was 95%. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. The pattern of growth observed for Clostridium botulinum type A1 strain ATCC 19397 is influenced by nutritional status and quorum sensing: a modelling perspective.

    Science.gov (United States)

    Ihekwaba, Adaoha E C; Mura, Ivan; Peck, Michael W; Barker, G C

    2015-12-01

    Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum are the most poisonous substances known to mankind. However, toxin regulation and signals triggering synthesis as well as the regulatory network and actors controlling toxin production are unknown. Experiments show that the neurotoxin gene is growth phase dependent for C. botulinum type A1 strain ATCC 19397, and toxin production is influenced both by culture conditions and nutritional status of the medium. Building mathematical models to describe the genetic and molecular machinery that drives the synthesis and release of BoNT requires a simultaneous description of the growth of the bacterium in culture. Here, we show four plausible modelling options which could be considered when constructing models describing the pattern of growth observed in a botulinum growth medium. Commonly used bacterial growth models are unsuitable to fit the pattern of growth observed, since they only include monotonic growth behaviour. We find that a model that includes both the nutritional status and the ability of the cells to sense their surroundings in a quorum-sensing manner is most successful at explaining the pattern of growth obtained for C. botulinum type A1 strain ATCC 19397. © FEMS 2015.

  7. Effect of Fill Temperature on Clostridium botulinum Type A Toxin Activity during the Hot Filling of Juice Bottles.

    Science.gov (United States)

    Skinner, Guy E; Fleischman, Gregory J; Balster, Fran; Reineke, Karl; Reddy, N Rukma; Larkin, John W

    2015-08-01

    The potential threat of terrorist attacks against the United States food supply using neurotoxin produced by Clostridium botulinum (BoNT) has resulted in the need for studying the effect of various food process operations on the bioavailability of this toxin. The objective of this study was to evaluate C. botulinum type A neurotoxin bioavailability after a simulated hot fill juice bottling operation. C. botulinum type A acid mud toxin (∼10(6) mouse lethal dose [MLD50]/ml) was deposited into juice bottles at an experimentally determined fastest cooling spot. Bottles (12 or 20 oz [355 and 592 ml]) were filled with either apple juice or an orange drink, at 80 or 85°C, in either upright or inverted orientations. Toxicity of the juice was evaluated as a function of holding time (1 to 2 min) by the mouse bioassay. The fastest cooling point in the upright orientation was determined to be at a bottle's bottom rim. In the inverted orientation, the fastest cooling point was in the bottle cap region. With respect to these two points, the upright bottle cooled faster than the inverted bottle, which was reflected in a higher inactivation of BoNT in the latter. For the orange drink (pH 2.9) toxicity was reduced by 0.5 × 10(6) MLD50/ml to a nondetectable level after 1 min in all bottle sizes, orientations, and temperatures as measured by the mouse bioassay. This indicates that there was at least a 0.5 × 10(6) MLD50/ml reduction in activity. Inactivation in apple juice (pH 4.0), to the same degree as in the orange drink, was found only for the inverted orientation at 85°C. Complete inactivation in apple juice for all conditions was found at a lower added toxin level of 0.25 × 10(5) MLD50/ml. In general, bottle inversion and filling at 85°C provided complete inactivation of BoNT to the 0.5 × 10(6) MLD50/ml level. All experiments resulted in the inactivation of 2.5 × 10(4) MLD50/ml of BoNT regardless of juice type, fill temperature, or bottle orientation and size.

  8. Structure of a Clostridium botulinum C143S thiaminase I/thiamin complex reveals active site architecture .

    Science.gov (United States)

    Sikowitz, Megan D; Shome, Brateen; Zhang, Yang; Begley, Tadhg P; Ealick, Steven E

    2013-11-05

    Thiaminases are responsible for the degradation of thiamin and its metabolites. Two classes of thiaminases have been identified based on their three-dimensional structures and their requirements for a nucleophilic second substrate. Although the reactions of several thiaminases have been characterized, the physiological role of thiamin degradation is not fully understood. We have determined the three-dimensional X-ray structure of an inactive C143S mutant of Clostridium botulinum (Cb) thiaminase I with bound thiamin at 2.2 Å resolution. The C143S/thiamin complex provides atomic level details of the orientation of thiamin upon binding to Cb-thiaminase I and the identity of active site residues involved in substrate binding and catalysis. The specific roles of active site residues were probed by using site directed mutagenesis and kinetic analyses, leading to a detailed mechanism for Cb-thiaminase I. The structure of Cb-thiaminase I is also compared to the functionally similar but structurally distinct thiaminase II.

  9. The Rho Termination Factor of Clostridium botulinum contains a Prion-Like Domain with a highly Amyloidogenic Core

    Directory of Open Access Journals (Sweden)

    Irantzu ePallares

    2016-01-01

    Full Text Available Prion-like proteins can switch between a soluble intrinsically disordered conformation and a highly ordered amyloid assembly. This conformational promiscuity is encoded in specific sequence regions, known as prion domains (PrDs. Prions are best known as the causative factors of neurological diseases in mammals. However, bioinformatics analyses reveal that proteins bearing PrDs are present in all kingdoms of life, including bacteria, thus supporting the idea that they serve conserved beneficial cellular functions. Despite the proportion of predicted prion-like proteins in bacterial proteomes is generally low, pathogenic species seem to have a higher prionic load, suggesting that these malleable proteins may favor pathogenic traits. In the present work, we performed a stringent computational analysis of the Clostridium botulinum pathogen proteome in the search for prion-like proteins. A total of 54 candidates were predicted for this anaerobic bacterium, including the transcription termination Rho factor. This RNA-binding protein has been shown to play a crucial role in bacterial adaptation to changing environments. We show here that the predicted disordered PrD domain of this RNA-binding protein contains an inner, highly polar, asparagine-rich short sequence able to spontaneously self-assemble into amyloid-like structures, bearing thus the potential to induce a Rho factor conformational switch that might rewire gene expression in response to environmental conditions.

  10. Botulinum toxin

    Directory of Open Access Journals (Sweden)

    Nigam P

    2010-01-01

    Full Text Available Botulinum toxin, one of the most poisonous biological substances known, is a neurotoxin produced by the bacterium Clostridium botulinum. C. botulinum elaborates eight antigenically distinguishable exotoxins (A, B, C 1 , C 2 , D, E, F and G. All serotypes interfere with neural transmission by blocking the release of acetylcholine, the principal neurotransmitter at the neuromuscular junction, causing muscle paralysis. The weakness induced by injection with botulinum toxin A usually lasts about three months. Botulinum toxins now play a very significant role in the management of a wide variety of medical conditions, especially strabismus and focal dystonias, hemifacial spasm, and various spastic movement disorders, headaches, hypersalivation, hyperhidrosis, and some chronic conditions that respond only partially to medical treatment. The list of possible new indications is rapidly expanding. The cosmetological applications include correction of lines, creases and wrinkling all over the face, chin, neck, and chest to dermatological applications such as hyperhidrosis. Injections with botulinum toxin are generally well tolerated and side effects are few. A precise knowledge and understanding of the functional anatomy of the mimetic muscles is absolutely necessary to correctly use botulinum toxins in clinical practice.

  11. Effect of Equilibrated pH and Indigenous Spoilage Microorganisms on the Inhibition of Proteolytic Clostridium botulinum Toxin Production in Experimental Meals under Temperature Abuse.

    Science.gov (United States)

    Golden, Max C; Wanless, Brandon J; David, Jairus R D; Lineback, D Scott; Talley, Ryan J; Kottapalli, Bala; Glass, Kathleen A

    2017-08-01

    Clostridium botulinum is a foreseeable biological hazard in prepared refrigerated meals that needs to be addressed in food safety plans. The objective of this study was to evaluate the effect of product composition and storage temperature on the inhibition of botulinum toxin formation in nine experimental meals (meat, vegetable, or carbohydrate based). Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin in samples stored at 25°C for up to 96 h for phase 1, or at 25°C for 12 h and then transferred to 12.5°C for up to 12 and 6 weeks in phases 1 and 2, respectively. For phase 1, none of the treatments (equilibrated pH 5.8) supported toxin production when stored at 25°C for 48 h, but toxin production was observed in all treatments at 72 h. For the remaining experiments with storage at 12.5°C, toxin production was dependent on equilibrated pH, storage time, and growth of indigenous spoilage microorganisms. In phase 1, no gross spoilage and no botulinum toxin was detected for any treatment (pH ≤5.8) stored at 12.5°C for 12 weeks. In phase 2, gross spoilage varied by commodity, with the brussels sprouts meal with pH 6.5 showing the most rapid spoilage within 2 weeks and botulinum toxin detected at 5 and 6 weeks for the control and cultured celery juice treatments, respectively. In contrast, spoilage microbes decreased the pH of a pH 5.9 beef treatment by 1.0 unit, potentially inhibiting C. botulinum through 6 weeks at 12.5°C. None of the other treatments with pH 5.8 or below supported toxin production or spoilage. This study provides validation for preventive controls in refrigerated meals. These include equilibrated product pH and storage temperature and time to inhibit toxin formation by proteolytic C. botulinum, but the impact of indigenous microflora on safety and interpretation of challenge studies is also highlighted.

  12. Neurotoxins from Clostridium botulinum (serotype A) isolated from the soil of Mendoza (Argentina) differ from the A-Hall archetype and from that causing infant botulism.

    Science.gov (United States)

    Caballero, P; Troncoso, M; Patterson, S I; López Gómez, C; Fernandez, R; Sosa, M A

    2016-10-01

    The type A of neurotoxin produced by Clostridium botulinum is the prevalent serotype in strains of Mendoza. The soil is the main reservoir for C.botulinum and is possibly one of the infection sources in infant botulism. In this study, we characterized and compared autochthonous C. botulinum strains and their neurotoxins. Bacterial samples were obtained from the soil and from fecal samples collected from children with infant botulism. We first observed differences in the appearance of the colonies between strains from each source and with the A Hall control strain. In addition, purified neurotoxins of both strains were found to be enriched in a band of 300 kDa, whereas the A-Hall strain was mainly made up of a band of ∼600 kDa. This finding is in line with the lack of hemagglutinating activity of the neurotoxins under study. Moreover, the proteolytic activity of C. botulinum neurotoxins was evaluated against SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins from rat brain. It was observed that both, SNAP 25 (synaptosomal-associated protein 25) and VAMP 2 (vesicle-associated membrane protein) were cleaved by the neurotoxins isolated from the soil strains, whereas the neurotoxins from infant botulism strains only induced a partial cleavage of VAMP 2. On the other hand, the neurotoxin from the A-Hall strain was able to cleave both proteins, though at a lesser extent. Our data indicate that the C.botulinum strain isolated from the soil, and its BoNT, exhibit different properties compared to the strain obtained from infant botulism patients, and from the A-Hall archetype. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Insights into the Origin of Clostridium botulinum Strains: Evolution of Distinct Restriction Endonuclease Sites in rrs (16S rRNA gene).

    Science.gov (United States)

    Bhushan, Ashish; Mukherjee, Tanmoy; Joshi, Jayadev; Shankar, Pratap; Kalia, Vipin Chandra

    2015-06-01

    Diversity analysis of Clostridium botulinum strains is complicated by high microheterogeneity caused by the presence of 9-22 copies of rrs (16S rRNA gene). The need is to mine genetic markers to identify very closely related strains. Multiple alignments of the nucleotide sequences of the 212 rrs of 13 C. botulinum strains revealed intra- and inter-genomic heterogeneity. Low intragenomic heterogeneity in rrs was evident in strains 230613, Alaska E43, Okra, Eklund 17B, Langeland, 657, Kyoto, BKT015925, and Loch Maree. The most heterogenous rrs sequences were those of C. botulinum strains ATCC 19397, Hall, H04402065, and ATCC 3502. In silico restriction mapping of these rrs sequences was observable with 137 type II Restriction endonucleases (REs). Nucleotide changes (NC) at these RE sites resulted in appearance of distinct and additional sites, and loss in certain others. De novo appearances of RE sites due to NC were recorded at different positions in rrs gene. A nucleotide transition A>G in rrs of C. botulinum Loch Maree and 657 resulted in the generation of 4 and 10 distinct RE sites, respectively. Transitions A>G, G>A, and T>C led to the loss of RE sites. A perusal of the entire NC and in silico RE mapping of rrs of C. botulinum strains provided insights into their evolution. Segregation of strains on the basis of RE digestion patterns of rrs was validated by the cladistic analysis involving six house keeping genes: dnaN, gyrB, metG, prfA, pyrG, and Rho.

  14. Non-linear pressure/temperature-dependence of high pressure thermal inactivation of proteolytic Clostridium botulinum type B in foods.

    Directory of Open Access Journals (Sweden)

    Maximilian B Maier

    Full Text Available The effect of high pressure thermal (HPT processing on the inactivation of spores of proteolytic type B Clostridium botulinum TMW 2.357 in four differently composed low-acid foods (green peas with ham, steamed sole, vegetable soup, braised veal was studied in an industrially feasible pressure range and temperatures between 100 and 120°C. Inactivation curves exhibited rapid inactivation during compression and decompression followed by strong tailing effects. The highest inactivation (approx. 6-log cycle reduction was obtained in braised veal at 600 MPa and 110°C after 300 s pressure-holding time. In general, inactivation curves exhibited similar negative exponential shapes, but maximum achievable inactivation levels were lower in foods with higher fat contents. At high treatment temperatures, spore inactivation was more effective at lower pressure levels (300 vs. 600 MPa, which indicates a non-linear pressure/temperature-dependence of the HPT spore inactivation efficiency. A comparison of spore inactivation levels achievable using HPT treatments versus a conventional heat sterilization treatment (121.1°C, 3 min illustrates the potential of combining high pressures and temperatures to replace conventional retorting with the possibility to reduce the process temperature or shorten the processing time. Finally, experiments using varying spore inoculation levels suggested the presence of a resistant fraction comprising approximately 0.01% of a spore population as reason for the pronounced tailing effects in survivor curves. The loss of the high resistance properties upon cultivation indicates that those differences develop during sporulation and are not linked to permanent modifications at the genetic level.

  15. Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

    Science.gov (United States)

    Baghban, Roghayyeh; Gargari, Seyed Latif Mousavi; Rajabibazl, Masoumeh; Nazarian, Shahram; Bakherad, Hamid

    2016-01-01

    Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  16. Growth of non-toxigenic Clostridium botulinum mutant LNT01 in cooked beef: One-step kinetic analysis and comparison with C. sporogenes and C. perfringens.

    Science.gov (United States)

    Huang, Lihan

    2018-05-01

    The objective of this study was to investigate the growth kinetics of Clostridium botulinum LNT01, a non-toxigenic mutant of C. botulinum 62A, in cooked ground beef. The spores of C. botulinum LNT01 were inoculated to ground beef and incubated anaerobically under different temperature conditions to observe growth and develop growth curves. A one-step kinetic analysis method was used to analyze the growth curves simultaneously to minimize the global residual error. The data analysis was performed using the USDA IPMP-Global Fit, with the Huang model as the primary model and the cardinal parameters model as the secondary model. The results of data analysis showed that the minimum, optimum, and maximum growth temperatures of this mutant are 11.5, 36.4, and 44.3 °C, and the estimated optimum specific growth rate is 0.633 ln CFU/g per h, or 0.275 log CFU/g per h. The maximum cell density is 7.84 log CFU/g. The models and kinetic parameters were validated using additional isothermal and dynamic growth curves. The resulting residual errors of validation followed a Laplace distribution, with about 60% of the residual errors within ±0.5 log CFU/g of experimental observations, suggesting that the models could predict the growth of C. botulinum LNT01 in ground beef with reasonable accuracy. Comparing with C. perfringens, C. botulinum LNT01 grows at much slower rates and with much longer lag times. Its growth kinetics is also very similar to C. sporogenes in ground beef. The results of computer simulation using kinetic models showed that, while prolific growth of C. perfringens may occur in ground beef during cooling, no growth of C. botulinum LNT01 or C. sporogenes would occur under the same cooling conditions. The models developed in this study may be used for prediction of the growth and risk assessments of proteolytic C. botulinum in cooked meats. Published by Elsevier Ltd.

  17. Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D and their mosaic variants C-D and D-C in a multicenter collaborative trial

    DEFF Research Database (Denmark)

    Woudstra, C.; Skarin, H.; Anniballi, F.

    2013-01-01

    Two real-time PCR arrays based on the GeneDisc® cycler platform (Pall-GeneDisc Technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated wi...

  18. Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D and their mosaic variants C-D and D-C in a multicenter collaborative trial

    NARCIS (Netherlands)

    Woudstra, C.; Skarin, H.; Anniballi, F.; Auricchio, B.; Medici, De D.; Bano, L.; Drigo, I.; Hansen, T.; Löfström, Ch.; Hamidjaja, R.; Rotterdam, van B.; Koene, M.G.J.

    2013-01-01

    Two real-time PCR arrays based on the GeneDisc(®) cycler platform (Pall-GeneDisc Technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated

  19. Effects of irradiation on growth and toxigenicity of Clostridium botulinum types A and B inoculated onto chicken skins

    International Nuclear Information System (INIS)

    Dezfulian, M.; Bartlett, J.G.

    1987-01-01

    This study was conducted to examine the effects of 0.3-Mrad irradiation on growth and toxigenicity of Clostridum botulinum types A and B on chicken skins. Irradiation followed by aerobic or anaerobic incubation at 30 0 C extended the shelf life of skin samples and delayed growth and toxin production by C. botulinum. During 2 weeks of incubation at 10 0 C, the irradiated and nonirradiated C. botulinum spores failed to grow or produce toxin

  20. 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)

    O'Flaherty, Sarah; Klaenhammer, Todd R

    2016-10-15

    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. 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 preferred over plasmid

  1. Esporos e toxinas de Clostridium botulinum dos tipos C e D em cacimbas no Vale do Araguaia, Goiás

    Directory of Open Access Journals (Sweden)

    Souza Aires M.

    2006-01-01

    Full Text Available Foram avaliadas a ocorrência e distribuição de esporos e toxinas de Clostridium botulinum tipos C e D em 300 cacimbas empregadas como bebedouro de bovinos em 130 propriedades rurais localizadas em 12 municípios do Vale do Araguaia, Estado de Goiás. A presença de esporos foi determinada indiretamente pelo cultivo em meio de cultura, seguido da inoculação e neutralização em camundongo das amostras de sedimento do interior das cacimbas, e do solo superficial e fezes de bovinos, coletadas ao seu redor. A presença de toxina foi avaliada diretamente pela inoculação em camundongo do sedimento filtrado das cacimbas, também seguida da neutralização em camundongo com antitoxinas C e D. A presença de esporos de C. botulinum foi significativamente maior (p<0,05 nas fezes de bovinos (31%, quando comparadas com os resultados das amostras de solo superficial (19% e dos sedimentos (10%. Foram detectadas toxinas botulínicas dos tipos C, D, ou classificadas como pertencentes ao complexo CD, em seis amostras (2% das 300 cacimbas. Das 130 propriedades trabalhadas, em 122(93,85% foram encontrados esporos ou toxinas de Clostridium botulinum em pelo menos uma das variáveis pesquisadas, enquanto somente 8(6,15% não apresentaram qualquer contaminação A idade e profundidade das cacimbas estiveram associadas com a freqüência de detecção de esporos e toxinas. Assim, quanto mais velhas e rasas, maior a freqüência do isolamento de esporos e toxinas. A contaminação das cacimbas do Vale do Araguaia goiano com esporos e toxinas do Clostridium botulinum tipos C e D demonstra o risco potencial permanente e crescente para a ocorrência da intoxicação botulínica de origem hídrica nos bovinos.

  2. Isolation of botulinolysin, a thiol-activated hemolysin, from serotype D Clostridium botulinum: A species-specific gene duplication in Clostridia.

    Science.gov (United States)

    Suzuki, Tomonori; Nagano, Thomas; Niwa, Koichi; Mutoh, Shingo; Uchino, Masataka; Tomizawa, Motohiro; Sagane, Yoshimasa; Watanabe, Toshihiro

    2016-12-01

    Botulinolysin (BLY) is a toxin produced by Clostridium botulinum that belongs to a group of thiol-activated hemolysins. In this study, a protein exhibiting hemolytic activity was purified from the culture supernatant of C. botulinum serotype D strain 4947. The purified protein displayed a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular mass of 55kDa, and its N-terminal and internal amino acid sequences exhibited high similarity to a group of thiol-activated hemolysins produced by gram-positive bacteria. Thus, the purified protein was identified as the BLY. Using the nucleotide sequences of previously cloned genes for hemolysins, two types of genes encoding BLY-like proteins were cloned unexpectedly. Molecular modeling analysis indicated that the products of both genes displayed very similar structures, despite the low sequence similarity. In silico screening revealed a specific duplication of the hemolysin gene restricted to serotypes C and D of C. botulinum and their related species among thiol-activated hemolysin-producing bacteria. Our findings provide important insights into the genetic characteristics of pathogenic bacteria. Copyright © 2016 Elsevier GmbH. All rights reserved.

  3. Linden flower (Tilia spp. as potential vehicle of Clostridium botulinum spores in the transmission of infant botulism El té de tilo como vehículo potencial de esporas de Clostridium botulinum en la transmisión del botulismo infantil

    Directory of Open Access Journals (Sweden)

    M. I. Bianco

    2009-12-01

    Full Text Available Infant botulism is an intestinal toxemia caused principally by Clostridium botulinum. Since the infection occurs in the intestinal tract, numerous food products have been investigated for the presence of C. botulinum and its neurotoxins. In many countries, people use linden flower (Tilia spp tea as a household remedy and give it to infants as a sedative. Therefore, to help provide a clear picture of this disease transmission, we investigated the presence of botulinum spores in linden flowers. In this study, we analyzed 100 samples of unwrapped linden flowers and 100 samples of linden flowers in tea bags to determine the prevalence and spore-load of C. botulinum. Results were analyzed by the Fisher test. We detected a prevalence of 3% of botulinum spores in the unwrapped linden flowers analyzed and a spore load of 30 spores per 100 grams. None of the industrialized linden flowers analyzed were contaminated with botulinum spores. C. botulinum type A was identified in two samples and type B in one sample. Linden flowers must be considered a potential vehicle of C. botulinum, and the ingestion of linden flower tea can represent a risk factor for infant botulism.El botulismo del lactante es una toxiinfección causada, principalmente, por Clostridium botulinum. Debido a que esta infección ocurre en el tracto intestinal, la presencia de esta bacteria y sus neurotoxinas ha sido investigada en numerosos alimentos. En muchos países se utiliza el té de tilo (Tilia spp. como sedante natural, el que se administra incluso a los lactantes. A fin de contribuir al esclarecimiento de la transmisión de esta enfermedad, se investigó la prevalencia y la carga de esporas botulínicas en esta hierba. Se analizaron 100 muestras de tilo comercializado a granel y 100 muestras de tilo industralizado en “saquitos”. Los resultados de prevalencia fueron analizados por el test de Fisher y la carga de esporas por la técnica del número más probable. Se halló una

  4. Alternative sigma factors SigF, SigE, and SigG are essential for sporulation in Clostridium botulinum ATCC 3502.

    Science.gov (United States)

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

    2014-08-01

    Clostridium botulinum produces heat-resistant endospores that may germinate and outgrow into neurotoxic cultures in foods. Sporulation is regulated by the transcription factor Spo0A and the alternative sigma factors SigF, SigE, SigG, and SigK in most spore formers studied to date. We constructed mutants of sigF, sigE, and sigG in C. botulinum ATCC 3502 and used quantitative reverse transcriptase PCR and electron microscopy to assess their expression of the sporulation pathway on transcriptional and morphological levels. In all three mutants the expression of spo0A was disrupted. The sigF and sigE mutants failed to induce sigG and sigK beyond exponential-phase levels and halted sporulation during asymmetric cell division. In the sigG mutant, peak transcription of sigE was delayed and sigK levels remained lower than that in the parent strain. The sigG mutant forespore was engulfed by the mother cell and possessed a spore coat but no peptidoglycan cortex. The findings suggest that SigF and SigE of C. botulinum ATCC 3502 are essential for early sporulation and late-stage induction of sigK, whereas SigG is essential for spore cortex formation but not for coat formation, as opposed to previous observations in B. subtilis sigG mutants. Our findings add to a growing body of evidence that regulation of sporulation in C. botulinum ATCC 3502, and among the clostridia, differs from the B. subtilis model. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Detection of Clostridium botulinum neurotoxin genes (A-F) in dairy farms from Northern Germany using PCR: A case-control study.

    Science.gov (United States)

    Fohler, Svenja; Discher, Sabrina; Jordan, Eva; Seyboldt, Christian; Klein, Guenter; Neubauer, Heinrich; Hoedemaker, Martina; Scheu, Theresa; Campe, Amely; Charlotte Jensen, Katharina; Abdulmawjood, Amir

    2016-06-01

    Classical botulism in cattle mainly occurs after ingestion of feed contaminated with preformed toxin. In 2001 a form of botulism ("visceral botulism") was postulated to occur after ingestion of Clostridium (C.) botulinum cells or spores, followed by colonization of the intestine, and local production of botulinum neurotoxin (BoNT) causing chronic generalized disease. To verify the potential role of C. botulinum in the described syndrome, a case-control study was conducted, including 139 farms. Fecal samples, rumen content, water and silage samples were collected on each farm. Real time BoNT gene PCR assays were conducted after enrichment in RCM (Reinforced Clostridial Medium) at 37 °C and conventional PCRs after enrichment in MCM (Modified Cooked Meat Medium) at 30 °C. Furthermore, a direct detection of BoNT genes without prior enrichment was attempted. BoNT A, B, C, D, E and F genes were detected in animal samples from 25 (17.99%), 3 (2.16%), 0 (0.0%), 2 (1.44%), 1 (0.72%), and 3 (2.16%) farms, respectively. Eleven feed samples were positive for BoNT A gene. By enrichment a significant increase in sensitivity was achieved. Therefore, this should be an essential part of any protocol. No significant differences regarding BoNT gene occurrence could be observed between Case and Control farms or chronically diseased and clinically healthy animals within the particular category. Thus, the postulated form of chronic botulism in cows could not be confirmed. This study supports the general opinion that C. botulinum can occasionally be found in the rumen and intestine of cows without causing disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Rational Design of Therapeutic and Diagnostic Against Botulinum Neurotoxin

    Science.gov (United States)

    2006-12-01

    Gram-positive bacilli bacterium, Clostridium botulinum, which can be found worldwide in soil. Clostridium tetani is a similar bacterium to C...B: Biological Sciences, 354, 259-268. 6. Nantel, A.J. (1999). Clostridium Botulinum. WHO international programme on chemical safety poisons...of Clostridium botulinum C1 neurotoxin. Nucleic Acids Research, 18, 4924. 20. Binz, T., Kurazono, H., Popoff, M.R., Eklund, M.W., Sakaguchi, G

  7. Optimal spore germination in Clostridium botulinum ATCC 3502 requires the presence of functional copies of SleB and YpeB, but not CwlJ.

    Science.gov (United States)

    Meaney, Carolyn A; Cartman, Stephen T; McClure, Peter J; Minton, Nigel P

    2015-08-01

    Germination, the process by which dormant endospores return to vegetative growth, is a critical process in the life cycle of the notorious pathogen Clostridium botulinum. Crucial is the degradation by hydrolytic enzymes of an inner peptidoglycan spore layer termed the cortex. Two mechanistically different systems of cortex lysis exist in spores of Clostridium species. C. botulinum ATCC 3502 harbours the Bacillus-like system of SleB, CwlJ and YpeB cortex lytic enzymes (CLEs). Through the construction of insertional gene knockout mutants in the sleB, cwlJ and ypeB genes of C. botulinum ATCC 3502 and the production of spores of each mutant strain, the effect on germination was assessed. This study demonstrates a reduced germination efficiency in spores carrying mutations in either sleB or ypeB with an approximate 2-fold reduction in heat resistant colony forming units (CFU/OD600) when plated on rich media. This reduction could be restored to wild-type levels by removing the spore coat and plating on media supplemented with lysozyme. It was observed that cwlJ spores displayed a similar germination efficiency as wild-type spores (P > 0.05). An optimal germinant commixture was identified to include a combination of l-alanine with sodium bicarbonate as it resulted in a 32% drop in OD600, while the additional incorporation of l-lactate resulted in a 57% decrease. Studies of the germination efficiency of spores prepared from all three CLE mutants was performed by monitoring the associated decrease in optical density but a germination defect was not observed in any of the CLE mutant strains. This was likely due to the lack of specificity of this particular assay. Taken together, these data indicate that functional copies of SleB and YpeB, but not CwlJ are required for the optimal germination of the spores of C. botulinum ATCC 3502. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Ultrastructure of a hexagonal array in exosporium of a highly sporogenic mutant of Clostridium botulinum type A revealed by electron microscopy using optical diffraction and filtration.

    Science.gov (United States)

    Masuda, K; Kawata, T; Takumi, K; Kinouchi, T

    1980-01-01

    The ultrastructure of a hexagonal array in the exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A strain 190L was studied by electron microscopy of negatively stained exosporium fragments using optical diffraction and filtration. The exosporium was composed of three or more lamellae showing and equilateral, hexagonal periodicity. Images of the single exosporium layer from which the noise had been filtered optically revealed that the hexagonally arranged, morphological unit of the exosporium was composed of three globular subunits about 2.1 nm in diameter which were arranged at the vertices of an equilateral triangle with sides of about 2.4 nm. The morphological units were arranged with a spacing of about 4.5 nm. the adjacent globular subunits appeared to be interconnected by delicate linkers.

  9. An electrochemiluminescence assay for the detection of bio threat agents in selected food matrices and in the screening of Clostridium botulinum outbreak strains associated with type A botulism.

    Science.gov (United States)

    Sachdeva, Amita; Singh, Ajay K; Sharma, Shashi K

    2014-03-15

    Specific screening methods for complex food matrices are needed that enable unambiguous and sensitive detection of bio threat agents (BTAs) such as Bacillus anthracis spores and microbial toxins (e.g. staphylococcal enterotoxin B (SEB) and clostridial botulinum neurotoxins (BoNTs)). The present study describes an image-based 96-well Meso Scale Discovery (MSD) electrochemiluminescence (ECL) assay for simultaneous detection of BTAs in dairy milk products. The limit of detection of this ECL assay is 40 pg mL⁻¹ for BoNT/A complex, 10 pg mL⁻¹ for SEB and 40000 CFU mL⁻¹ for Bacillus anthracis spores in dairy milk products. The ECL assay was successfully applied to screen type A Clostridium botulinum outbreak strains. The results of the study indicate that this ECL assay is very sensitive, rapid (<6 h) and multiplex in nature. The ECL assay has potential for use as an in vitro screening method for BTAs over other comparable immunoassays. © 2013 Society of Chemical Industry.

  10. The variability of times to detect growth from individual Clostridium botulinum type E endospores is differentially affected by high pressure treatments

    Science.gov (United States)

    Lenz, Christian A.; Schnabel, Juliane; Vogel, Rudi F.

    2014-10-01

    High pressure thermal (HPT) processing is a candidate technology for the production of safe and stable food. However, little is known about the effect of HPT or high hydrostatic pressure (HHP) treatments at ambient temperature on the variability of times to detect growth from individual spores. We investigated this effect by treating Clostridium botulinum type E spores with HHP (200-600 MPa, 20°C) and HPT (600 MPa, 80°C and 800 MPa, 60°C). Our results indicate that the mean detection times increase and the frequency distribution shifts toward longer times when HHP treatment intensity is increased. HPT treatments result in a highly scattered distribution. In contrast, pressure levels ≤300 MPa decrease detection times and heterogeneity of their distribution, which could lead to an increase in the potential risk originating from C. botulinum type E spores. Data provided here could help to refine risk assessment regarding this important food intoxicator.

  11. Clostridium botulinum Toxin Production in Relation to Spoilage of Atlantic Salmon (Salmo salar) Packaged in Films of Varying Oxygen Permeabilities and with Different Atmospheres.

    Science.gov (United States)

    Erickson, Marilyn C; Ma, Li M; Doyle, Michael P

    2015-11-01

    Shelf life of fish packaged under modified atmosphere (MA) is extended, but within the United States, commercial application of MA with impermeable packaging films is restricted due to concerns that botulinum toxin production would precede spoilage when contaminated fish are held at abusive storage temperatures. Use of semipermeable packaging films has been advocated; however, previous studies are inconclusive in determining the oxygen transmission rate (OTR) of a film that is needed to achieve an acceptable margin of safety (i.e., toxin production occurs only after spoilage). This study was conducted to determine the influence of OTR (target OTRs of 3 to 15,000) on the development of spoilage volatiles and toxin in salmon inoculated with type E Clostridium botulinum and subjected to air, vacuum, or 75:25 CO2:N2 MA and storage temperatures of 4, 8, 12, or 16°C. The most dominant headspace volatile peak that was produced during spoilage of samples at 4, 8 or 12°C was a peak, having a Kovats retention index (KI) of 753, and at which external standards of 2- or 3-methyl 1-butanol also eluted. Under anaerobic conditions, both the aerobic microbial populations and the size of the KI 753 spoilage peak were less in inoculated samples compared with uninoculated samples. C. botulinum-inoculated samples that were stored at 12 or 16°C under conditions favorable for anaerobic growth were also characterized by a KI 688 peak. Using a previously developed model that related the percentage of elderly consumers who would prepare a sample having the KI 753 spoilage peak of a specific size, it was determined that for salmon packaged with 3 or 3,000 OTR films under any atmosphere and stored at 12 or 16°C, 2 to 61% of the consumers could potentially prepare toxin-contaminated samples. Hence, when abusive storage conditions are suspected, the fish should not be consumed.

  12. Structural constraints-based evaluation of immunogenic avirulent toxins from Clostridium botulinum C2 and C3 toxins as subunit vaccines.

    Science.gov (United States)

    Prisilla, A; Prathiviraj, R; Sasikala, R; Chellapandi, P

    2016-10-01

    Clostridium botulinum (group-III) is an anaerobic bacterium producing C2 and C3 toxins in addition to botulinum neurotoxins in avian and mammalian cells. C2 and C3 toxins are members of bacterial ADP-ribosyltransferase superfamily, which modify the eukaryotic cell surface proteins by ADP-ribosylation reaction. Herein, the mutant proteins with lack of catalytic and pore forming function derived from C2 (C2I and C2II) and C3 toxins were computationally evaluated to understand their structure-function integrity. We have chosen many structural constraints including local structural environment, folding process, backbone conformation, conformational dynamic sub-space, NAD-binding specificity and antigenic determinants for screening of suitable avirulent toxins. A total of 20 avirulent mutants were identified out of 23 mutants, which were experimentally produced by site-directed mutagenesis. No changes in secondary structural elements in particular to α-helices and β-sheets and also in fold rate of all-β classes. Structural stability was maintained by reordered hydrophobic and hydrogen bonding patterns. Molecular dynamic studies suggested that coupled mutations may restrain the binding affinity to NAD(+) or protein substrate upon structural destabilization. Avirulent toxins of this study have stable energetic backbone conformation with a common blue print of folding process. Molecular docking studies revealed that avirulent mutants formed more favorable hydrogen bonding with the side-chain of amino acids near to conserved NAD-binding core, despite of restraining NAD-binding specificity. Thus, structural constraints in the avirulent toxins would determine their immunogenic nature for the prioritization of protein-based subunit vaccine/immunogens to avian and veterinary animals infected with C. botulinum. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium botulinum strain PS-5

    Science.gov (United States)

    Botulinum neurotoxins (BoNTs) are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins (NAPs). The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presenc...

  14. Clostridium botulinum type D/C intoxication in a dairy cow stock in Saxony-Anhalt (Germany)--report on an innovative diagnostic approach.

    Science.gov (United States)

    Dlabola, Janine; Hashish, Emad; Pauly, Birgit; Kubisiak, Bernd; Behm, Ingrid; Heseler, Rüdiger; Schliephake, Annette; Wieler, Lothar H; Neubauer, Heinrich; Seyboldt, Christian

    2016-01-01

    Botulism in cattle is a rare but serious disease. In Germany there is no obligation to report botulism in animals and therefore a precise morbidity rate is not available. In this manuscript we describe an outbreak of Clostridium (C.) botulinum neurotoxin (BoNT) intoxication in a Saxony-Anhalt dairy cow stock of 286 Holstein-Friesian cows and offspring in spring/summer 2009 and its diagnostic approach. 122 animals showed clinical signs of BoNT intoxication. 115 of the affected animals (40.2% of the herd) independent of age died or had to be euthanized. Therapeutic attempts failed in almost all diseased cows, only four calves and three heifers recovered. Diagnostic samples of several animals (n = 4) (liver, ruminal and intestinal contents) and feed (n = 6) were tested for BoNT genes by polymerase chain reaction (PCR). BoNT gene type D was found in several (n = 8) organ samples. The PCR results allowed a preselection of samples for BoNT that were then tested by the mouse bioassay. Thus, the number of mice being inoculated in the mouse bioassay could be reduced. The mouse bioassay turned out positive (wasp-waist) in three preselected organ samples and the neutralization test of one sample with type-specific antitoxin confirmed the presence of BoNT type D. We succeeded in isolating a C. botulinum strain from a liver sample which was typed as a D/C mosaic strain by sequence analysis of the toxin gene. However, the source of the BoNT intoxication could not be traced back.

  15. Perfil sorológico das amostras de Clostridium botulinum tipos C e D utilizadas para produção de imunógenos no Brasil

    Directory of Open Access Journals (Sweden)

    Almeida A.C.

    2000-01-01

    Full Text Available Com o objetivo de avaliar o perfil sorológico de três amostras de Clostridium botulinum tipo C e três do tipo D utilizadas para produção de imunógenos no Brasil, determinou-se o índice de eficiência e o grau de homologia sorológica dentro de cada tipo. O índice de eficiência mostrou a mesma tendência para os dois tipos. Os consumos relativos de antitoxinas foram proporcionais nos níveis de 20, 200 e 1000 DL50, verificados através de curvas de neutralização que apresentaram inclinações semelhantes. A quantificação da variação do consumo de antitoxinas expresso em grau de homologia sorológica reflete uma similaridade relativa entre as amostras, sendo neutralizados 80 a 91,5% de determinantes antigênicos para o tipo C e 85 a 95% para o tipo D. Pelos resultados apresentados, as amostras analisadas comportaram-se como variantes sorológicas nos níveis de testes de 20, 200 e 1000 DL50.

  16. High resolution crystal structures of the receptor-binding domain of Clostridium botulinum neurotoxin serotypes A and FA

    Directory of Open Access Journals (Sweden)

    Jonathan R. Davies

    2018-03-01

    Full Text Available The binding specificity of botulinum neurotoxins (BoNTs is primarily a consequence of their ability to bind to multiple receptors at the same time. BoNTs consist of three distinct domains, a metalloprotease light chain (LC, a translocation domain (HN and a receptor-binding domain (HC. Here we report the crystal structure of HC/FA, complementing an existing structure through the modelling of a previously unresolved loop which is important for receptor-binding. Our HC/FA structure also contains a previously unidentified disulphide bond, which we have also observed in one of two crystal forms of HC/A1. This may have implications for receptor-binding and future recombinant toxin production.

  17. H-NOX from Clostridium botulinum, like H-NOX from Thermoanaerobacter tengcongensis, Binds Oxygen but with a Less Stable Oxyferrous Heme Intermediate.

    Science.gov (United States)

    Wu, Gang; Liu, Wen; Berka, Vladimir; Tsai, Ah-Lim

    2015-12-08

    Heme nitric oxide/oxygen binding protein isolated from the obligate anaerobe Clostridium botulinum (Cb H-NOX) was previously reported to bind NO with a femtomolar K(D) (Nioche, P. et al. Science 2004, 306, 1550-1553). On the other hand, no oxyferrous Cb H-NOX was observed despite full conservation of the key residues that stabilize the oxyferrous complex in the H-NOX from Thermoanaerobacter tengcongensis (Tt H-NOX) (the same study). In this study, we re-measured the kinetics/affinities of Cb H-NOX for CO, NO, and O2. K(D)(CO) for the simple one-step equilibrium binding was 1.6 × 10(-7) M. The K(D)(NO) of Cb H-NOX was 8.0 × 10(-11) M for the first six-coordinate NO complex, and the previous femtomolar K(D)(NO) was actually an apparent K(D) for its multiple-step NO binding. An oxyferrous Cb H-NOX was clearly observed with a K(D)(O2) of 5.3 × 10(-5) M, which is significantly higher than Tt H-NOX's K(D)(O2) = 4.4 × 10(-8) M. The gaseous ligand binding of Cb H-NOX provides another supportive example for the "sliding scale rule" hypothesis (Tsai, A.-L. et al. Antioxid. Redox Signal. 2012, 17, 1246-1263), and the presence of hydrogen bond donor Tyr139 in Cb H-NOX selectively enhanced its affinity for oxygen.

  18. Structures of Clostridium Botulinum Neurotoxin Serotype A Light Chain Complexed with Small-Molecule Inhibitors Highlight Active-Site Flexibility

    Energy Technology Data Exchange (ETDEWEB)

    Silvaggi,N.; Boldt, G.; Hixon, M.; Kennedy, J.; Tzipori, S.; Janda, K.; Allen, K.

    2007-01-01

    The potential for the use of Clostridial neurotoxins as bioweapons makes the development of small-molecule inhibitors of these deadly toxins a top priority. Recently, screening of a random hydroxamate library identified a small-molecule inhibitor of C. botulinum Neurotoxin Serotype A Light Chain (BoNT/A-LC), 4-chlorocinnamic hydroxamate, a derivative of which has been shown to have in vivo efficacy in mice and no toxicity. We describe the X-ray crystal structures of BoNT/A-LC in complexes with two potent small-molecule inhibitors. The structures of the enzyme with 4-chlorocinnamic hydroxamate or 2,4-dichlorocinnamic hydroxamate bound are compared to the structure of the enzyme complexed with L-arginine hydroxamate, an inhibitor with modest affinity. Taken together, this suite of structures provides surprising insights into the BoNT/A-LC active site, including unexpected conformational flexibility at the S1' site that changes the electrostatic environment of the binding pocket. Information gained from these structures will inform the design and optimization of more effective small-molecule inhibitors of BoNT/A-LC.

  19. Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D and their mosaic variants C-D and D-C in a multicenter collaborative trial.

    Science.gov (United States)

    Woudstra, Cedric; Skarin, Hanna; Anniballi, Fabrizio; Auricchio, Bruna; De Medici, Dario; Bano, Luca; Drigo, Ilenia; Hansen, Trine; Löfström, Charlotta; Hamidjaja, Raditijo; van Rotterdam, Bart J; Koene, Miriam; Bäyon-Auboyer, Marie-Hélène; Buffereau, Jean-Philippe; Fach, Patrick

    2013-08-01

    Two real-time PCR arrays based on the GeneDisc(®) cycler platform (Pall-GeneDisc Technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated with avian and mammalian botulism. The GeneDisc(®) arrays developed as part of the DG Home funded European project 'AnibioThreat' were highly sensitive and specific when tested on pure isolates and naturally contaminated samples (mostly clinical specimen from avian origin). Results of the multicenter collaborative trial involving eight laboratories in five European Countries (two laboratories in France, Italy and The Netherlands, one laboratory in Denmark and Sweden), using DNA extracts issued from 33 pure isolates and 48 naturally contaminated samples associated with animal botulism cases, demonstrated the robustness of these tests. Results showed a concordance among the eight laboratories of 99.4%-100% for both arrays. The reproducibility of the tests was high with a relative standard deviation ranging from 1.1% to 7.1%. Considering the high level of agreement achieved between the laboratories these PCR arrays constitute robust and suitable tools for rapid detection of C. botulinum types C, D and mosaic types C-D and D-C. These are the first tests for C. botulinum C and D that have been evaluated in a European multicenter collaborative trial. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Inactivation of bacterial spores by combination processes: ultraviolet plus gamma radiation. [Streptococcus faecium, micrococcus radiodurans, clostridium botulinum

    Energy Technology Data Exchange (ETDEWEB)

    Grecz, N.; Durban, E.

    1973-01-01

    Bacterial spores, viruses and some vegetative bacteria such as Streptococcus faecium and Micrococcus radiodurans are distinguished by high radiation resistance. In order to lay a theoretical basis for biomedical sterilization applications, we have investigated the combined action of uv and gamma rays. Spores of two strains of C. botulinum were selected, a highly radiation resistant strain, 33A having a D/sub 10/-value of 0.32 Mrad, and a relatively radiation sensitive strain, 51B having a D/sub 10/-value of 0.12 Mrad. Strain 33A exhibits an extensive initial ''shoulder'' in its uv as well as gamma ray survival curves; strain 51B shows only a slight shoulder. The shoulder in the gamma ray survival curve of spores of strain 33A could be reduced or completely eliminated by preirradiation with uv. Simultaneously the D/sub 10/-value for gamma inactivation of spores of 33A was reduced substantially. For example, the gamma resistance was reduced almost to half of its original D/sub 10/-value by uv-preirradiation for only one minute under an 8 watt GE germicidal lamp. The effect of uv-preirradiation on the radiation sensitive strain 51B was less pronounced. In fact, there was about seven fold higher positive interaction (synergism) between uv and gamma radiation in 33A spores than in 51B spores. The experiments suggest that interference with DNA repair enzymes in the radiation resistant strain are responsible for lethal synergism between uv and gamma radiation. A hypothesis is developed attempting to explain the combined effect of these two radiations in terms of a special summation of known DNA lesions in the cell. These observations emphasize the potential practical advantages of combining uv and gamma rays for effective sterilization of certain biomedical devices, drugs and biologicals.

  1. Substrate Binding Mode and Its Implication on Drug Design for Botulinum Neurotoxin A

    National Research Council Canada - National Science Library

    Kumaran, Desigan; Rawat, Richa; Ahmed, S. A; Swaminathan, Subramanyam

    2008-01-01

    The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins, the causative agents of botulism, block the neurotransmitter release by specifically cleaving one of the three SNARE...

  2. On botulinum neurotoxin variability.

    Science.gov (United States)

    Montecucco, Cesare; Rasotto, Maria Berica

    2015-01-06

    The rapidly growing number of botulinum neurotoxin sequences poses the problem of the possible evolutionary significance of the variability of these superpotent neurotoxins for toxin-producing Clostridium species. To progress in the understanding of this remarkable phenomenon, we suggest that researchers should (i) abandon an anthropocentric view of these neurotoxins as human botulism-causing agents or as human therapeutics, (ii) begin to investigate in depth the role of botulinum neurotoxins in animal botulism in the wilderness, and (iii) devote large efforts to next-generation sequencing of soil samples to identify novel botulinum neurotoxins. In order to compare the fitness of the different toxins, we suggest that assays of all the steps from toxin production to animal death should be performed. Copyright © 2015 Montecucco and Rasotto.

  3. Ocorrência e distribuição de esporos de Clostridium botulinum tipos C e D em áreas de criação de búfalos na Baixada Maranhense

    Directory of Open Access Journals (Sweden)

    Silva Tânia M.D.

    1998-01-01

    Full Text Available Botulismo é enzoótico na criação de búfalos da Baixada Maranhense, Estado do Maranhão. No presente trabalho foram realizados estudos para verificar a ocorrência e distribuição de esporos de Clostridium botulinum tipos C e D em amostras de solo, limo e fezes de búfalos, colhidas aleatoriamente em áreas inundáveis da criação de búfalos nessa Baixada. A evidenciação de esporos foi realizada em 40 amostras de fezes, 65 de limo e 35 de solo, provenientes de quatro municípios, pelo cultivo em meio de cultura com carne cozida e posterior inoculação do sobrenadante filtrado em camundongo, na tentativa de verificação da presença de toxina botulínica. A tipificação de amostras positivas foi realizada pela microfixação de complemento. Os resultados revelaram que 104 (74,28% das 140 amostras examinadas foram positivas para a presença de esporos de C. botulinum pelo teste indireto. Não houve diferença significativa (P>0,05 entre os valores obtidos quando das análises das amostras de solo (77,1%, limo (60,0% e fezes (95,0%. Das 28 amostras de solo, limo e fezes positivas, que foram utilizadas para a tipificação, quatro (14,29% foram classificadas como tipo C, 23 (82,14% como tipo D e uma (3,5% como pertencente ao complexo CD. Os resultados revelaram uma alta contaminação ambiental por C. botulinum em áreas de criação de búfalos da Baixada Maranhen-se. A identificação de outros tipos e de subtipos de C. botulinum não foi realizada.

  4. Analysis of the neurotoxin complex genes in Clostridium botulinum A1-A4 and B1 strains: BoNT/A3, /Ba4 and /B1 clusters are located within plasmids.

    Directory of Open Access Journals (Sweden)

    Theresa J Smith

    Full Text Available BACKGROUND: Clostridium botulinum and related clostridial species express extremely potent neurotoxins known as botulinum neurotoxins (BoNTs that cause long-lasting, potentially fatal intoxications in humans and other mammals. The amino acid variation within the BoNT is used to categorize the species into seven immunologically distinct BoNT serotypes (A-G which are further divided into subtypes. The BoNTs are located within two generally conserved gene arrangements known as botulinum progenitor complexes which encode toxin-associated proteins involved in toxin stability and expression. METHODOLOGY/PRINCIPAL FINDINGS: Because serotype A and B strains are responsible for the vast majority of human botulism cases worldwide, the location, arrangement and sequences of genes from eight different toxin complexes representing four different BoNT/A subtypes (BoNT/A1-Ba4 and one BoNT/B1 strain were examined. The bivalent Ba4 strain contained both the BoNT/A4 and BoNT/bvB toxin clusters. The arrangements of the BoNT/A3 and BoNT/A4 subtypes differed from the BoNT/A1 strains and were similar to those of BoNT/A2. However, unlike the BoNT/A2 subtype, the toxin complex genes of BoNT/A3 and BoNT/A4 were found within large plasmids and not within the chromosome. In the Ba4 strain, both BoNT toxin clusters (A4 and bivalent B were located within the same 270 kb plasmid, separated by 97 kb. Complete genomic sequencing of the BoNT/B1 strain also revealed that its toxin complex genes were located within a 149 kb plasmid and the BoNT/A3 complex is within a 267 kb plasmid. CONCLUSIONS/SIGNIFICANCE: Despite their size differences and the BoNT genes they contain, the three plasmids containing these toxin cluster genes share significant sequence identity. The presence of partial insertion sequence (IS elements, evidence of recombination/gene duplication events, and the discovery of the BoNT/A3, BoNT/Ba4 and BoNT/B1 toxin complex genes within plasmids illustrate the

  5. Development of a quail embryo model for the detection of botulinum toxin type A activity

    Science.gov (United States)

    Clostridium botulinum is a ubiquitous microorganism which under certain anaerobic conditions can produce botulinum toxins. Due to concerns in regards to both food-borne illness and the potential use of botulinum toxin as a biological weapon, the capability to assess the amount of toxin in a food or...

  6. Development of a quail embryo model for the detection of botulinum neurotoxin activity

    Science.gov (United States)

    Clostridium botulinum is a ubiquitous microorganism that under anaerobic conditions produces botulinum neurotoxins. In regards to both food-borne illness and the potential use of botulinum toxin as a biological weapon, the capability to assess the amount of toxin in a food or environmental sample e...

  7. Botulinum Toxin for Rhinitis.

    Science.gov (United States)

    Ozcan, Cengiz; Ismi, Onur

    2016-08-01

    Rhinitis is a common clinical entity. Besides nasal obstruction, itching, and sneezing, one of the most important symptoms of rhinitis is nasal hypersecretion produced by nasal glands and exudate from the nasal vascular bed. Allergic rhinitis is an IgE-mediated inflammatory reaction of nasal mucosa after exposure to environmental allergens. Idiopathic rhinitis describes rhinitis symptoms that occur after non-allergic, noninfectious irritants. Specific allergen avoidance, topical nasal decongestants, nasal corticosteroids, immunotherapy, and sinonasal surgery are the main treatment options. Because the current treatment modalities are not enough for reducing rhinorrhea in some patients, novel treatment options are required to solve this problem. Botulinum toxin is an exotoxin generated by Clostridium botulinum. It disturbs the signal transmission at the neuromuscular and neuroglandular junction by inhibiting the acetylcholine release from the presynaptic nerve terminal. It has been widely used in neuromuscular, hypersecretory, and autonomic nerve system disorders. There have been a lot of published articles concerning the effect of this toxin on rhinitis symptoms. Based on the results of these reports, intranasal botulinum toxin A administration appears to be a safe and effective treatment method for decreasing rhinitis symptoms in rhinitis patients with a long-lasting effect. Botulinum toxin type A will be a good treatment option for the chronic rhinitis patients who are resistant to other treatment methods.

  8. MOLECULAR-BIOLOGY OF CLOSTRIDIAL TOXINS - EXPRESSION OF MESSENGER-RNAS ENCODING TETANUS AND BOTULINUM NEUROTOXINS IN APLYSIA NEURONS

    NARCIS (Netherlands)

    MOCHIDA, S; POULAIN, B; EISEL, U; BINZ, T; KURAZONO, H; NIEMANN, H; TAUC, L

    1990-01-01

    mRNAs encoding the light chain of tetanus and botulinum neurotoxins were transcribed, in vitro, from the cloned and specifically truncated genes of Clostridium tetani and Clostridium botulinum, respectively, and injected into presynaptic identified cholinergic neurons of the buccal ganglia of

  9. A cell-permeable fusion protein based on Clostridium botulinum C2 toxin for delivery of p53 tumorsuppressor into cancer cells.

    Directory of Open Access Journals (Sweden)

    Jörg Fahrer

    Full Text Available Genetically engineered bacterial protein toxins are attractive systems for delivery of exogenous proteins into the cytosol of mammalian cells. The binary C2 toxin from C. botulinum has emerged as powerful delivery vehicle, which rests on its binding/translocation component C2IIa and the genetically modified adaptor domain C2IN that act in concert to trigger cell uptake. The p53 tumor suppressor protein has a crucial function in suppressing carcinogenesis and is frequently inactivated by diverse mechanisms in human tumor cells. Therefore, we constructed a C2IN-p53 fusion protein, which is internalized into cancer cells by C2IIa. To this end, the C2IN-p53 fusion construct was overexpressed in E. coli with good solubility, purified by heparin affinity chromatography and protein identity was confirmed by immunoblotting. We demonstrated that the fusion protein is capable of binding to the p53 consensus-DNA with high affinity in a p53-specific manner in vitro. Next, the internalization of C2IN-p53 was monitored in HeLa cells by cell fractionation and immunoblot analysis, which revealed a C2IIa-mediated translocation of the fusion protein into the cytosol. The uptake was also shown in A549 and Saos-2 cells with similar efficiency. These findings were further corroborated by confocal immunofluorescence analyses of C2IN-p53/C2IIa-treated HeLa and A549 cells, displaying predominantly cytoplasmic localization of the fusion construct.

  10. [Simplified preliminary identification of some species of Clostridium].

    Science.gov (United States)

    Rivas, M; Cinto, R O; Frade, A H

    1982-01-01

    A dichotomous key is proposed for the identification of eight species of Clostridium: Clostridium botulinum, C. butyricum, C. haemolyticum, C. histolyticum, C. paraperfringens, C. perfringens, C. sporogenes y C. subterminale, on the basis of the Gram staining, catalase production, growth on nutrient agar, glucose utilization, motility test, gelatin hydrolysis, lecithinase production, human blood hemolysis and the test of mice toxicity.

  11. Rapid multiplex immunoassay to distinguish botulinum neurotoxin serotypes on a single lateral flow device(Abstract)

    Science.gov (United States)

    Clostridium botulinum produces seven antigenically distinct serotypes of botulinum neurotoxin (BoNT/A–G). The potency of these toxins result in a high mortality rate with BoNT/A and /B accounting for most of the naturally occurring outbreaks. The ease of BoNT production and their potential use as bi...

  12. Lipid diversity among botulinum neurotoxin-producing clostridia

    Science.gov (United States)

    Guan, Ziqiang; Johnston, Norah C.; Raetz, Christian R. H.; Johnson, Eric A.

    2012-01-01

    Clostridium botulinum has been classified into four groupings (groups I to IV) based on physiological characteristics and 16S rRNA sequencing. We have examined the lipid compositions of 11 representative strains of C. botulinum and a strain of Clostridium sporogenes by 2D-TLC and by MS. All strains contained phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylethanolamine (PE) in both the all-acyl and the alk-1′-enyl (plasmalogen) forms. Five strains in proteolytic group I, which are related to C. sporogenes, contained varying amounts of an ethanolamine-phosphate derivative of N-acetylglucosaminyl-diradylglycerol, which is also present in C. sporogenes. Three strains in group II, which are related to Clostridium butyricum, Clostridium beijerinckii and Clostridium acetobutylicum, contained lipids characteristic of these saccharolytic species: a glycerol acetal and a PG acetal of the plasmalogen form of PE. Two group III strains, which are related to Clostridium novyi, contained amino-acyl derivatives of PG, which are also found in C. novyi. A strain in group IV had PE, PG and CL, but none of the distinguishing lipids. This work shows that the lipidome of C. botulinum is consistent with its classification by other methods. PMID:22837302

  13. Botulinum Toxin

    Science.gov (United States)

    2009-01-01

    show inconsistent and erroneous results. In vivo experiments using the rat extensor digitorum longus (EDL) muscle assay showed sensitivities of rat... digitorum longus muscle paralyzed by local injection of botulinum neurotoxin. Toxicon 34: 237--49. Amon, S. (1995). Botulism as an intestinal...BoNT -induced paralysis. References Adler, M., MacDonald, D.A., Sellin, L.C., Parker, G.W. (1996). Effect of 3,4-diaminopyridine on rat extensor

  14. ClosTron-mediated engineering of Clostridium.

    Science.gov (United States)

    Kuehne, Sarah A; Heap, John T; Cooksley, Clare M; Cartman, Stephen T; Minton, Nigel P

    2011-01-01

    The genus Clostridium is a diverse assemblage of Gram positive, anaerobic, endospore-forming bacteria. Whilst certain species have achieved notoriety as important animal and human pathogens (e.g. Clostridium difficile, Clostridium botulinum, Clostridium tetani, and Clostridium perfringens), the vast majority of the genus are entirely benign, and are able to undertake all manner of useful biotransformations. Prominent amongst them are those species able to produce the biofuels, butanol and ethanol from biomass-derived residues, such as Clostridium acetobutylicum, Clostridium beijerinkii, Clostridium thermocellum, and Clostridium phytofermentans. The prominence of the genus in disease and biotechnology has led to the need for more effective means of genetic modification. The historical absence of methods based on conventional strategies for "knock-in" and "knock-out" in Clostridium has led to the adoption of recombination-independent procedures, typified by ClosTron technology. The ClosTron uses a retargeted group II intron and a retro-transposition-activated marker to selectively insert DNA into defined sites within the genome, to bring about gene inactivation and/or cargo DNA delivery. The procedure is extremely efficient, rapid, and requires minimal effort by the operator.

  15. [Botulinum neurotoxin].

    Science.gov (United States)

    Poulain, B

    2010-01-01

    Botulinum toxin is a multi-molecular complex comprised of a neuro-active moiety (i.e. botulinum neurotoxin) and several associated non-toxic proteins. The toxin dissociates rapidly at plasmatic pH, thereby releasing neurotoxin. Nerve terminals only take up the neurotoxin. In the peripheral nerve system, the neurotoxin mainly blocks acetylcholine release. When acting at the neuromuscular junctions, this results in paralysis of the muscle fibers. The duration of the neurotoxin action is mainly determined by the life-time of neurotoxin molecules inside the nerve terminals. Inhibition of cholinergic transmission induces rapid atrophy of the muscle fibres, and, sometimes, sprouting from poisoned nerve terminals. These effects, as well as the acetylcholine release blockade are entirely reversible. When injected in the periphery, a direct action of botulinum neurotoxin in the central nervous system remains unlikely despite its retrograde ascent demonstrated in animal models. However, indirect effects are numerous. The constituting proteins of the toxin complex can lead to immunisation against the non-toxic associated proteins and neurotoxin. Only the antibodies directed against neurotoxin are potentially neutralizing. Copyright 2009 Elsevier Masson SAS. All rights reserved.

  16. Preterm Infant-Associated Clostridium tertium, Clostridium cadaveris, and Clostridium paraputrificum Strains: Genomic and Evolutionary Insights.

    Science.gov (United States)

    Kiu, Raymond; Caim, Shabhonam; Alcon-Giner, Cristina; Belteki, Gusztav; Clarke, Paul; Pickard, Derek; Dougan, Gordon; Hall, Lindsay J

    2017-10-01

    Clostridium species (particularly Clostridium difficile, Clostridium botulinum, Clostridium tetani and Clostridium perfringens) are associated with a range of human and animal diseases. Several other species including Clostridium tertium, Clostridium cadaveris, and Clostridium paraputrificum have also been linked with sporadic human infections, however there is very limited, or in some cases, no genomic information publicly available. Thus, we isolated one C. tertium strain, one C. cadaveris strain and three C. paraputrificum strains from preterm infants residing within neonatal intensive care units and performed Whole Genome Sequencing (WGS) using Illumina HiSeq. In this report, we announce the open availability of the draft genomes: C. tertium LH009, C. cadaveris LH052, C. paraputrificum LH025, C. paraputrificum LH058, and C. paraputrificum LH141. These genomes were checked for contamination in silico to ensure purity, and we confirmed species identity and phylogeny using both 16S rRNA gene sequences (from PCR and in silico) and WGS-based approaches. Average Nucleotide Identity (ANI) was used to differentiate genomes from their closest relatives to further confirm speciation boundaries. We also analysed the genomes for virulence-related factors and antimicrobial resistance genes, and detected presence of tetracycline and methicillin resistance, and potentially harmful enzymes, including multiple phospholipases and toxins. The availability of genomic data in open databases, in tandem with our initial insights into the genomic content and virulence traits of these pathogenic Clostridium species, should enable the scientific community to further investigate the disease-causing mechanisms of these bacteria with a view to enhancing clinical diagnosis and treatment. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  17. Finished Whole-Genome Sequences ofClostridium butyricumToxin Subtype E4 andClostridium baratiiToxin Subtype F7 Strains.

    Science.gov (United States)

    Halpin, Jessica L; Hill, Karen; Johnson, Shannon L; Bruce, David Carlton; Shirey, T Brian; Dykes, Janet K; Lúquez, Carolina

    2017-07-20

    Clostridium butyricum and Clostridium baratii species have been known to produce botulinum toxin types E and F, respectively, which can cause botulism, a rare but serious neuroparalytic disease. Here, we present finished genome sequences for two of these clinically relevant strains. Copyright © 2017 Halpin et al.

  18. Phylogenetic positions of Clostridium novyi and Clostridium haemolyticum based on 16S rDNA sequences.

    Science.gov (United States)

    Sasaki, Y; Takikawa, N; Kojima, A; Norimatsu, M; Suzuki, S; Tamura, Y

    2001-05-01

    The partial sequences (1465 bp) of the 16S rDNA of Clostridium novyi types A, B and C and Clostridium haemolyticum were determined. C. novyi types A, B and C and C. haemolyticum clustered with Clostridium botulinum types C and D. Moreover, the 16S rDNA sequences of C. novyi type B strains and C. haemolyticum strains were completely identical; they differed by 1 bp (level of similarity > 99.9%) from that of C. novyi type C, they were 98.7% homologous to that of C. novyi type A (relative positions 28-1520 of the Escherichia coli 16S rDNA sequence) and they exhibited a higher similarity to the 16S rDNA sequence of C. botulinum types D and C than to that of C. novyi type A. These results suggest that C. novyi types B and C and C. haemolyticum may be one independent species generated from the same phylogenetic origin.

  19. Mathematical modeling and growth kinetics of Clostridium sporogenes in cooked beef

    Science.gov (United States)

    Clostridium sporogenes PA 3679 is a common surrogate for proteolytic Clostridium botulinum for thermal process development and validation. However, little information is available concerning the growth kinetics of C. sporogenes in food. Therefore, the objective of this study was to investigate the...

  20. Comparative pathogenomics of Clostridium tetani.

    Directory of Open Access Journals (Sweden)

    Jonathan E Cohen

    Full Text Available Clostridium tetani and Clostridium botulinum produce two of the most potent neurotoxins known, tetanus neurotoxin and botulinum neurotoxin, respectively. Extensive biochemical and genetic investigation has been devoted to identifying and characterizing various C. botulinum strains. Less effort has been focused on studying C. tetani likely because recently sequenced strains of C. tetani show much less genetic diversity than C. botulinum strains and because widespread vaccination efforts have reduced the public health threat from tetanus. Our aim was to acquire genomic data on the U.S. vaccine strain of C. tetani to better understand its genetic relationship to previously published genomic data from European vaccine strains. We performed high throughput genomic sequence analysis on two wild-type and two vaccine C. tetani strains. Comparative genomic analysis was performed using these and previously published genomic data for seven other C. tetani strains. Our analysis focused on single nucleotide polymorphisms (SNP and four distinct constituents of the mobile genome (mobilome: a hypervariable flagellar glycosylation island region, five conserved bacteriophage insertion regions, variations in three CRISPR (clustered regularly interspaced short palindromic repeats-Cas (CRISPR-associated systems, and a single plasmid. Intact type IA and IB CRISPR/Cas systems were within 10 of 11 strains. A type IIIA CRISPR/Cas system was present in two strains. Phage infection histories derived from CRISPR-Cas sequences indicate C. tetani encounters phages common among commensal gut bacteria and soil-borne organisms consistent with C. tetani distribution in nature. All vaccine strains form a clade distinct from currently sequenced wild type strains when considering variations in these mobile elements. SNP, flagellar glycosylation island, prophage content and CRISPR/Cas phylogenic histories provide tentative evidence suggesting vaccine and wild type strains share a

  1. Comparative pathogenomics of Clostridium tetani.

    Science.gov (United States)

    Cohen, Jonathan E; Wang, Rong; Shen, Rong-Fong; Wu, Wells W; Keller, James E

    2017-01-01

    Clostridium tetani and Clostridium botulinum produce two of the most potent neurotoxins known, tetanus neurotoxin and botulinum neurotoxin, respectively. Extensive biochemical and genetic investigation has been devoted to identifying and characterizing various C. botulinum strains. Less effort has been focused on studying C. tetani likely because recently sequenced strains of C. tetani show much less genetic diversity than C. botulinum strains and because widespread vaccination efforts have reduced the public health threat from tetanus. Our aim was to acquire genomic data on the U.S. vaccine strain of C. tetani to better understand its genetic relationship to previously published genomic data from European vaccine strains. We performed high throughput genomic sequence analysis on two wild-type and two vaccine C. tetani strains. Comparative genomic analysis was performed using these and previously published genomic data for seven other C. tetani strains. Our analysis focused on single nucleotide polymorphisms (SNP) and four distinct constituents of the mobile genome (mobilome): a hypervariable flagellar glycosylation island region, five conserved bacteriophage insertion regions, variations in three CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems, and a single plasmid. Intact type IA and IB CRISPR/Cas systems were within 10 of 11 strains. A type IIIA CRISPR/Cas system was present in two strains. Phage infection histories derived from CRISPR-Cas sequences indicate C. tetani encounters phages common among commensal gut bacteria and soil-borne organisms consistent with C. tetani distribution in nature. All vaccine strains form a clade distinct from currently sequenced wild type strains when considering variations in these mobile elements. SNP, flagellar glycosylation island, prophage content and CRISPR/Cas phylogenic histories provide tentative evidence suggesting vaccine and wild type strains share a common ancestor.

  2. Evaluation of a Botulinum Toxoid, Type B, for the Prevention of Shaker Foal Syndrome,

    Science.gov (United States)

    1981-11-06

    pregnancy has been shown to be an efficient means of passively immunizing foals against the tetanus neurotoxin produced by Clostridium tetani . The...9,12 Clostridium botulinum, type B, has been isolated repeatedly from the feces of affected foals 4 and the disease can be reproduced experimentally...immunization of their dams with aluminum hydroxide-adsorbed tetanus toxoid (derived from the neurotoxin of C. tetani ) given in 2-ml doses at 2 months

  3. Application of Botulinum toxin Type A: An arsenal in dentistry

    Directory of Open Access Journals (Sweden)

    Lakshmana B Rao

    2011-01-01

    Full Text Available An extremely effective way of preventing damage to and enhancing treatment of dental hard tissues and restorations would be to ′′de-programme′′ the muscles responsible for excessive destructive forces and other gnathological-related diseases. The new paradigm is the intramuscular injection of Botulinum toxin type A (BOTOX into the affected muscles. It is a natural protein produced by anaerobic bacterium, Clostridium botulinum. The toxin inhibits the release of acetylcholine (ACH, a neurotransmitter responsible for the activation of muscle contraction and glandular secretion, and its administration results in reduction of tone in the injected muscle. There are seven distinct serotypes of Botulinum toxin, viz., A, B, C, D, E, F, and G, which differ in their potency, duration of action, and cellular target sites. This paper describes the different applications of BOTOX in dentistry.

  4. Botulinum toxin for masseter hypertrophy.

    Science.gov (United States)

    Fedorowicz, Zbys; van Zuuren, Esther J; Schoones, Jan

    2013-09-09

    Benign masseter muscle hypertrophy is an uncommon clinical phenomenon of uncertain aetiology which is characterised by a soft swelling near the angle of the mandible. The swelling may on occasion be associated with facial pain and can be prominent enough to be considered cosmetically disfiguring. Varying degrees of success have been reported for some of the treatment options for masseter hypertrophy, which range from simple pharmacotherapy to more invasive surgical reduction. Injection of botulinum toxin type A into the masseter muscle is generally considered a less invasive modality and has been advocated for cosmetic sculpting of the lower face. Botulinum toxin type A is a powerful neurotoxin which is produced by the anaerobic organism Clostridium botulinum and when injected into a muscle causes interference with the neurotransmitter mechanism producing selective paralysis and subsequent atrophy of the muscle.This review is an update of a previously published Cochrane review. To assess the efficacy and safety of botulinum toxin type A compared to placebo or no treatment, for the management of benign bilateral masseter hypertrophy. We searched the following databases from inception to April 2013: the Cochrane Central Register of Controlled Trials (CENTRAL); MEDLINE (via PubMed); EMBASE (via embase.com); Web of Science; CINAHL; Academic Search Premier (via EBSCOhost); ScienceDirect; LILACS (via BIREME); PubMed Central and Google Scholar (from 1700 to 19 April 2013). We searched two bibliographic databases of regional journals (IndMED and Iranmedex) which were expected to contain relevant trials. We also searched reference lists of relevant articles and contacted investigators to identify additional published and unpublished studies. Randomised controlled trials (RCTs) and controlled clinical trials (CCTs) comparing intra-masseteric injections of botulinum toxin versus placebo administered for cosmetic facial sculpting in individuals of any age with bilateral benign

  5. Botulinum toxin A for trismus in cephalic tetanus

    Directory of Open Access Journals (Sweden)

    Luiz Augusto F. Andrade

    1994-09-01

    Full Text Available Cephalic tetanus is a localized form of tetanus. As in generalized forms , trismus is a prominent feature of the disease, leading to considerable difficulty in feeding, swallowing of the saliva and mouth hygiene. These difficulties often precede respiratory problems and aspiration bronchopneumonia is a frequent life-threatening complication. Muscle relaxants other than curare drugs may show a limited benefit for relieving trismus. Tetanospasmin, the tetanic neurotoxin, and botulinum toxin share many similarities, having a closely related chemical structure, an origin from related microorganisms (Clostridium tetani and Clostridium botulinum, respectively, and presumably, the same mechanisms of action in the neuron. The difference between the two lies in their peculiar neurospecificity, acting in different neurons. Injection of minute doses of botulinum toxin in the muscles involved in focal dystonias or other localized spastic disorders have proved to be very effective in these conditions. We describe the use of botulinum toxin A in the successful treatment of trismus in a patient suffering from cephalic tetanus. We believe that this form of treatment may be of value in lowering the risk of pulmonary complications in tetanic patients.

  6. A Potent Peptidomimetic Inhibitor of Botulinum Neurotoxin Serotype A has a Very Different Conformation than SNAP-25 Substrate

    Science.gov (United States)

    2008-10-07

    inhibit other BoNT serotypes or thermolysin . 15. SUBJECT TERMS Clostridium botulinum, neurotoxin, serotype A, peptidomimetic inhibitor , light chain...20 0 BoNT/E SNAP-25, residues 1–206 20 0 BoNT/F VAMP residues 1–94 20 0 Thermolysin Synthetic peptidea 500 0 The inhibitor concentration was 4 mM. a...Structure ArticleA Potent Peptidomimetic Inhibitor of Botulinum Neurotoxin Serotype A Has a Very Different Conformation than SNAP-25 Substrate Jorge

  7. Characterization of Hemagglutinin Negative Botulinum Progenitor Toxins

    Directory of Open Access Journals (Sweden)

    Suzanne R. Kalb

    2017-06-01

    Full Text Available Botulism is a disease involving intoxication with botulinum neurotoxins (BoNTs, toxic proteins produced by Clostridium botulinum and other clostridia. The 150 kDa neurotoxin is produced in conjunction with other proteins to form the botulinum progenitor toxin complex (PTC, alternating in size from 300 kDa to 500 kDa. These progenitor complexes can be classified into hemagglutinin positive or hemagglutinin negative, depending on the ability of some of the neurotoxin-associated proteins (NAPs to cause hemagglutination. The hemagglutinin positive progenitor toxin complex consists of BoNT, nontoxic non-hemagglutinin (NTNH, and three hemagglutinin proteins; HA-70, HA-33, and HA-17. Hemagglutinin negative progenitor toxin complexes contain BoNT and NTNH as the minimally functional PTC (M-PTC, but not the three hemagglutinin proteins. Interestingly, the genome of hemagglutinin negative progenitor toxin complexes comprises open reading frames (orfs which encode for three proteins, but the existence of these proteins has not yet been extensively demonstrated. In this work, we demonstrate that these three proteins exist and form part of the PTC for hemagglutinin negative complexes. Several hemagglutinin negative strains producing BoNT/A, /E, and /F were found to contain the three open reading frame proteins. Additionally, several BoNT/A-containing bivalent strains were examined, and NAPs from both genes, including the open reading frame proteins, were associated with BoNT/A. The open reading frame encoded proteins are more easily removed from the botulinum complex than the hemagglutinin proteins, but are present in several BoNT/A and /F toxin preparations. These are not easily removed from the BoNT/E complex, however, and are present even in commercially-available purified BoNT/E complex.

  8. Botulinum Neurotoxin Injections

    Science.gov (United States)

    ... Special Events Faces of Dystonia Donate Donate Online Membership Find an Event Donor Bill of Rights About Dystonia Symptoms & Diagnosis Forms of Dystonia Genetics Glossary Treatment Find a Doctor Oral Medications Botulinum Neurotoxin Neurosurgery ...

  9. Complementation of a Clostridium perfringens spo0A mutant with wild-type spo0A from other Clostridium species.

    Science.gov (United States)

    Huang, I-Hsiu; Sarker, Mahfuzur R

    2006-09-01

    To evaluate whether C. perfringens can be used as a model organism for studying the sporulation process in other clostridia, C. perfringens spo0A mutant IH101 was complemented with wild-type spo0A from four different Clostridium species. Wild-type spo0A from C. acetobutylicum or C. tetani, but not from C. botulinum or C. difficile, restored sporulation and enterotoxin production in IH101. The ability of spo0A from C. botulinum or C. difficile to complement the lack of spore formation in IH101 might be due, at least in part, to the low levels of spo0A transcription and Spo0A production.

  10. Beneficial and harmful roles of bacteria from the Clostridium genus.

    Science.gov (United States)

    Samul, Dorota; Worsztynowicz, Paulina; Leja, Katarzyna; Grajek, Włodzimierz

    2013-01-01

    Bacteria of the Clostridium genus are often described only as a biological threat and a foe of mankind. However, many of them have positive properties and thanks to them they may be used in many industry branches (e.g., in solvents and alcohol production, in medicine, and also in esthetic cosmetology). During the last 10 years interest in application of C. botulinum and C. tetani in medicine significantly increased. Currently, the structure and biochemical properties of neurotoxins produced by these bacterial species, as well as possibilities of application of such toxins as botulinum as a therapeutic factor in humans, are being intensely researched. The main aim of this article is to demonstrate that bacteria from Clostridium spp. are not only pathogens and the enemy of humanity but they also have many important beneficial properties which make them usable among many chemical, medical, and cosmetic applications.

  11. Botulinum Neurotoxin Is Shielded by NTNHA in an Interlocked Complex

    Energy Technology Data Exchange (ETDEWEB)

    Gu, Shenyan; Rumpel, Sophie; Zhou, Jie; Strotmeier, Jasmin; Bigalke, Hans; Perry, Kay; Shoemaker, Charles B.; Rummel, Andreas; Jin, Rongsheng (Cornell); (Tufts); (Hannover-MED); (Sanford-Burnham)

    2012-03-28

    Botulinum neurotoxins (BoNTs) are highly poisonous substances that are also effective medicines. Accidental BoNT poisoning often occurs through ingestion of Clostridium botulinum-contaminated food. Here, we present the crystal structure of a BoNT in complex with a clostridial nontoxic nonhemagglutinin (NTNHA) protein at 2.7 angstroms. Biochemical and functional studies show that NTNHA provides large and multivalent binding interfaces to protect BoNT from gastrointestinal degradation. Moreover, the structure highlights key residues in BoNT that regulate complex assembly in a pH-dependent manner. Collectively, our findings define the molecular mechanisms by which NTNHA shields BoNT in the hostile gastrointestinal environment and releases it upon entry into the circulation. These results will assist in the design of small molecules for inhibiting oral BoNT intoxication and of delivery vehicles for oral administration of biologics.

  12. Notice of CDC's discontinuation of investigational pentavalent (ABCDE) botulinum toxoid vaccine for workers at risk for occupational exposure to botulinum toxins.

    Science.gov (United States)

    2011-10-28

    Effective November 30, 2011, CDC will no longer provide investigational pentavalent (ABCDE) botulinum toxoid (PBT) for vaccination of workers at risk for occupational exposure to botulinum serotypes A, B, C, D, and E. This change might affect persons working in public health laboratories, research facilities, and manufacturing institutions who work with botulinum toxin or neurotoxin-producing species of Clostridium. CDC's decision is based on an assessment of the available data, which indicate a decline in immunogenicity of some of the toxin serotypes. The occurrence of moderate local reactions related to annual booster doses also has increased, which was noted in the 1990s at the U.S. Army Medical Research Institute for Infectious Diseases and resulted in a change in boosting from an annual requirement to only boosting when antibody titers have declined significantly. Additionally, the PBT was manufactured more than 30 years ago. CDC, therefore, has decided not to continue offering this investigational product.

  13. Tetanus: Pathophysiology, Treatment, and the Possibility of Using Botulinum Toxin against Tetanus-Induced Rigidity and Spasms

    Directory of Open Access Journals (Sweden)

    Bjørnar Hassel

    2013-01-01

    Full Text Available Tetanus toxin, the product of Clostridium tetani, is the cause of tetanus symptoms. Tetanus toxin is taken up into terminals of lower motor neurons and transported axonally to the spinal cord and/or brainstem. Here the toxin moves trans-synaptically into inhibitory nerve terminals, where vesicular release of inhibitory neurotransmitters becomes blocked, leading to disinhibition of lower motor neurons. Muscle rigidity and spasms ensue, often manifesting as trismus/lockjaw, dysphagia, opistotonus, or rigidity and spasms of respiratory, laryngeal, and abdominal muscles, which may cause respiratory failure. Botulinum toxin, in contrast, largely remains in lower motor neuron terminals, inhibiting acetylcholine release and muscle activity. Therefore, botulinum toxin may reduce tetanus symptoms. Trismus may be treated with botulinum toxin injections into the masseter and temporalis muscles. This should probably be done early in the course of tetanus to reduce the risk of pulmonary aspiration, involuntary tongue biting, anorexia and dental caries. Other muscle groups are also amenable to botulinum toxin treatment. Six tetanus patients have been successfully treated with botulinum toxin A. This review discusses the use of botulinum toxin for tetanus in the context of the pathophysiology, symptomatology, and medical treatment of Clostridium tetani infection.

  14. Tetanus: Pathophysiology, Treatment, and the Possibility of Using Botulinum Toxin against Tetanus-Induced Rigidity and Spasms

    Science.gov (United States)

    Hassel, Bjørnar

    2013-01-01

    Tetanus toxin, the product of Clostridium tetani, is the cause of tetanus symptoms. Tetanus toxin is taken up into terminals of lower motor neurons and transported axonally to the spinal cord and/or brainstem. Here the toxin moves trans-synaptically into inhibitory nerve terminals, where vesicular release of inhibitory neurotransmitters becomes blocked, leading to disinhibition of lower motor neurons. Muscle rigidity and spasms ensue, often manifesting as trismus/lockjaw, dysphagia, opistotonus, or rigidity and spasms of respiratory, laryngeal, and abdominal muscles, which may cause respiratory failure. Botulinum toxin, in contrast, largely remains in lower motor neuron terminals, inhibiting acetylcholine release and muscle activity. Therefore, botulinum toxin may reduce tetanus symptoms. Trismus may be treated with botulinum toxin injections into the masseter and temporalis muscles. This should probably be done early in the course of tetanus to reduce the risk of pulmonary aspiration, involuntary tongue biting, anorexia and dental caries. Other muscle groups are also amenable to botulinum toxin treatment. Six tetanus patients have been successfully treated with botulinum toxin A. This review discusses the use of botulinum toxin for tetanus in the context of the pathophysiology, symptomatology, and medical treatment of Clostridium tetani infection. PMID:23299659

  15. Emerging treatments for overactive bladder: clinical potential of botulinum toxins

    Directory of Open Access Journals (Sweden)

    Tincello DG

    2014-05-01

    Full Text Available Douglas G Tincello,1,2 Tina Rashid,2 Vladimir Revicky21Reproductive Sciences Section, Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK; 2Urogynecology Unit, Women's and Children's Clinical Business Unit, University Hospitals of Leicester National Health Service Trust, Leicester, UKAbstract: Overactive bladder (OAB is a symptom syndrome including urgency, frequency, and nocturia – with or without incontinence. It is a common manifestation of detrusor overactivity (DO. DO is a urodynamic observation of spontaneous or provoked contractions of the detrusor muscle is seen during the filling phase of the micturition cycle. OAB is, therefore, both a motor and sensory disorder. Botulinum toxin is a purified form of the neurotoxin from Clostridium botulinum and has been used in medicine for many years. Over the last 10 years, it has been used for the treatment of DO and OAB when standard treatments, such as bladder training and oral anticholinergic medication, have failed to provide symptom relief. Botulinum toxin acts by irreversibly preventing neurotransmitter release from the neurons in the motor end plate and also at sensory synapses, although the clinical effect is not permanent due to the growth of new connections within treated tissues. It is known that botulinum toxin modulates vanillioid, purinergic, capsaicin, and muscarinic receptor expression within the lamina propria, returning them to levels seen in normal bladders. Clinically, the effect of botulinum toxin on symptoms of OAB and DO is profound, with large effects upon the symptom of urgency, and also large effects on frequency, nocturia, leakage episodes, and continence rates. These effects have been seen consistently within eight randomized trials and numerous case series. Botulinum toxin appears safe, with the only common side effect being that of voiding difficulty, occurring in up to 10% of treated patients. Dosing regimens are variable, depending on

  16. Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature

    Science.gov (United States)

    2016-08-26

    Legeay, C. Bouchier, P. Bouvet, M.R. Popoff, An atypical outbreak of food-borne botulism due to Clostridium botulinum types B and E from ham , J Clin...07755 infant botulism/New York , 2004 AFD33678 B8 Maehongson foodborne botulism/Thailand, 2010 AFN61309 BoNT/C C1 Stockholm mink/Sweden BAA14235 CD...6813 soil/Maryland BAA08418 BoNT/D D 1873 ham /Chad, 1958 EES90380 DC VPI 5995 South Africa ABP48747 BoNT/E E1 Beluga whale/Alaska, 1952 CAA43999

  17. Successful use of botulinum toxin a in intractable, severe muscle spasms in spinal cord injury: A case report

    Directory of Open Access Journals (Sweden)

    Madhuri A Lokapur

    2017-01-01

    Full Text Available Botulinum toxin is a protein produced by Clostridium botulinum, which inhibits muscle contraction by transiently blocking the release of acetylcholine at the neuromuscular junction. At a neuromuscular junction, the toxin inactivates some of the fusion proteins, such as SNAP-25, syntaxin, or synaptobrevin, which are essential for cellular function. This process involves the temporary inhibition of presynaptic acetylcholine release; consequently, its effects are restricted to motor neurons that depend on the cholinergic transmission (muscular plate, gland innervating cells. Injections of botulinum toxin A have been shown to be useful in the treatment of etiologically diverse types of muscle spasms. Ultrasonography (USG has been used as a guide for confirming muscle fasciculations and also is an effective tool for confirming precise needle positioning and correct drug placement. We describe a case of a 25-year-old man with meningomyelocele and paraparesis with painful muscle spasms in bilateral thighs treated by USG-guided botulinum toxin injection.

  18. The Mechanism of the Beneficial Effect of Botulinum Toxin Type a Used in the Treatment of Temporomandibular Joints Dysfunction.

    Science.gov (United States)

    Malgorzata, Pihut; Piotr, Ceranowicz; Edward, Kijak

    2017-01-01

    In the course of temporomandibular joint, dysfunctions very often occur to the excessive increase in tension of masticatory muscles, so the main aim of the treatment is reduction of this hypertension of muscles. For this reason, we use botulinum toxin type A, which is produced by Grampositive Clostridium bacteria. There are six serotypes of the toxin: A, B, C, D, E, F, and G. The botulinum toxin type A was first isolated in 1920s. Today, botulinum toxin type A is used increasingly more often as an efficient and patient-friendly therapy in neurology, ophthalmology, neurology, urology and laryngology. The aim of the article was to review the literature and description of the current knowledge concerned with mechanism of action of botulinum toxin type A, clinical applications and metabolic determinants of muscle contraction and the beneficial effect of this drug on the state of muscle tension. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay.

    Science.gov (United States)

    Williamson, Charles H D; Vazquez, Adam J; Hill, Karen; Smith, Theresa J; Nottingham, Roxanne; Stone, Nathan E; Sobek, Colin J; Cocking, Jill H; Fernández, Rafael A; Caballero, Patricia A; Leiser, Owen P; Keim, Paul; Sahl, Jason W

    2017-09-15

    Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes , and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present ( ha positive [ ha + ] or orfX + ). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene ( bont ) clusters. IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes , and two major subgroups within C. botulinum group II. Since Bo

  20. Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novel sigma factor.

    Science.gov (United States)

    Dupuy, Bruno; Mani, Nagraj; Katayama, Seiichi; Sonenshein, Abraham L

    2005-02-01

    Expression of the plasmid-encoded Clostridium perfringens gene for bacteriocin BCN5 was shown to depend in vivo and in vitro on the activity of UviA protein. UviA, also plasmid-encoded, proved to be an RNA polymerase sigma factor and was also partly autoregulatory. The uviA gene has two promoters; one provided a UviA-independent, basal level of gene expression while the stronger, UviA-dependent promoter was only utilized after the cell experienced DNA damage. As a result, BCN5 synthesis is induced by treatment with UV light or mitomycin C. UviA is related to a special class of sigma factors found to date only in Clostridium species and responsible for activating transcription of toxin genes in Clostridium difficile, Clostridium tetani, and Clostridium botulinum.

  1. Antipruritic effects of botulinum neurotoxins

    DEFF Research Database (Denmark)

    Gazerani, Parisa

    2018-01-01

    This review explores current evidence to demonstrate that botulinum neurotoxins (BoNTs) exert antipruritic effects. Both experimental and clinical conditions in which botulinum neurotoxins have been applied for pruritus relief will be presented and significant findings will be highlighted. Potent....... Potential mechanisms underlying antipruritic effects will also be discussed and ongoing challenges and unmet needs will be addressed.......This review explores current evidence to demonstrate that botulinum neurotoxins (BoNTs) exert antipruritic effects. Both experimental and clinical conditions in which botulinum neurotoxins have been applied for pruritus relief will be presented and significant findings will be highlighted...

  2. Universal and specific quantitative detection of botulinum neurotoxin genes

    Directory of Open Access Journals (Sweden)

    Arnon Stephen S

    2010-10-01

    Full Text Available Abstract Background Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces seven antigenic variants of botulinum toxin that are distinguished serologically and termed "serotypes". Botulinum toxin blocks the release of acetylcholine at neuromuscular junctions resulting in flaccid paralysis. The potential lethality of the disease warrants a fast and accurate means of diagnosing suspected instances of food contamination or human intoxication. Currently, the Food and Drug Administration (FDA-accepted assay to detect and type botulinum neurotoxins (BoNTs is the mouse protection bioassay. While specific and sensitive, this assay requires the use of laboratory animals, may take up to four days to achieve a diagnosis, and is unsuitable for high-throughput analysis. We report here a two-step PCR assay that identifies all toxin types, that achieves the specificity of the mouse bioassay while surpassing it in equivalent sensitivity, that has capability for high-throughput analysis, and that provides quantitative results within hours. The first step of our assay consists of a conventional PCR that detects the presence of C. botulinum regardless of the neurotoxin type. The second step uses quantitative PCR (qPCR technology to determine the specific serotype of the neurotoxin. Results We assayed purified C. botulinum DNA and crude toxin preparations, as well as food and stool from healthy individuals spiked with purified BoNT DNA, and one stool sample from a case of infant botulism for the presence of the NTNH gene, which is part of the BoNT gene cluster, and for the presence of serotype-specific BoNT genes. The PCR surpassed the mouse bioassay both in specificity and sensitivity, detecting positive signals in BoNT preparations containing well below the 1 LD50 required for detection via the mouse bioassay. These results were type-specific and we were reliably able to quantify as few as 10 genomic copies. Conclusions While other studies

  3. Small RNAs in the genus Clostridium.

    Science.gov (United States)

    Chen, Yili; Indurthi, Dinesh C; Jones, Shawn W; Papoutsakis, Eleftherios T

    2011-01-25

    The genus Clostridium includes major human pathogens and species important to cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are emerging as crucial regulatory molecules in all organisms, but they have not been investigated in clostridia. Research on sRNAs in clostridia is hindered by the absence of a systematic method to identify sRNA candidates, thus delegating clostridial sRNA research to a hit-and-miss process. Thus, we wanted to develop a method to identify potential sRNAs in the Clostridium genus to open up the field of sRNA research in clostridia. Using comparative genomics analyses combined with predictions of rho-independent terminators and promoters, we predicted sRNAs in 21 clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, C. botulinum (eight strains), C. cellulolyticum, C. difficile, C. kluyveri (two strains), C. novyi, C. perfringens (three strains), C. phytofermentans, C. tetani, and C. thermocellum. Although more than one-third of predicted sRNAs have Shine-Dalgarno (SD) sequences, only one-sixth have a start codon downstream of SD sequences; thus, most of the predicted sRNAs are noncoding RNAs. Quantitative reverse transcription-PCR (Q-RT-PCR) and Northern analysis were employed to test the presence of a randomly chosen set of sRNAs in C. acetobutylicum and several C. botulinum strains, leading to the confirmation of a large fraction of the tested sRNAs. We identified a conserved, novel sRNA which, together with the downstream gene coding for an ATP-binding cassette (ABC) transporter gene, responds to the antibiotic clindamycin. The number of predicted sRNAs correlated with the physiological function of the species (high for pathogens, low for cellulolytic, and intermediate for solventogenic), but not with 16S rRNA-based phylogeny. Copyright © 2011 Chen et al.

  4. New Elements To Consider When Modeling the Hazards Associated with Botulinum Neurotoxin in Food.

    Science.gov (United States)

    Ihekwaba, Adaoha E C; Mura, Ivan; Malakar, Pradeep K; Walshaw, John; Peck, Michael W; Barker, G C

    2016-01-15

    Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum are the most potent biological substances known to mankind. BoNTs are the agents responsible for botulism, a rare condition affecting the neuromuscular junction and causing a spectrum of diseases ranging from mild cranial nerve palsies to acute respiratory failure and death. BoNTs are a potential biowarfare threat and a public health hazard, since outbreaks of foodborne botulism are caused by the ingestion of preformed BoNTs in food. Currently, mathematical models relating to the hazards associated with C. botulinum, which are largely empirical, make major contributions to botulinum risk assessment. Evaluated using statistical techniques, these models simulate the response of the bacterium to environmental conditions. Though empirical models have been successfully incorporated into risk assessments to support food safety decision making, this process includes significant uncertainties so that relevant decision making is frequently conservative and inflexible. Progression involves encoding into the models cellular processes at a molecular level, especially the details of the genetic and molecular machinery. This addition drives the connection between biological mechanisms and botulism risk assessment and hazard management strategies. This review brings together elements currently described in the literature that will be useful in building quantitative models of C. botulinum neurotoxin production. Subsequently, it outlines how the established form of modeling could be extended to include these new elements. Ultimately, this can offer further contributions to risk assessments to support food safety decision making. Copyright © 2015 Ihekwaba et al.

  5. Genetic Diversity Among Botulinum Neurotoxin Producing Clostridial Strains

    Energy Technology Data Exchange (ETDEWEB)

    Hill, K K; Smith, T J; Helma, C H; Ticknor, L O; Foley, B T; Svennson, R T; Brown, J L; Johnson, E A; Smith, L A; Okinaka, R T; Jackson, P J; Marks, J D

    2006-07-06

    Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore forming rod-shaped bacteria which have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and even death in humans and various other animal species. A collection of 174 C. botulinum strains were examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT A-G). Analysis of the16S rRNA sequences confirmed earlier reports of at least four distinct genomic backgrounds (Groups I-IV) each of which has independently acquired one or more BoNT serotypes through horizontal gene transfer. AFLP analysis provided higher resolution, and can be used to further subdivide the four groups into sub-groups. Sequencing of the BoNT genes from serotypes A, B and E in multiple strains confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes, and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven serotypes of BoNT were compared and show varying degrees of interrelatedness and recombination as has been previously noted for the NTNH gene which is linked to BoNT. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for treatment of botulism.

  6. USE OF BOTULINUM TOXIN TYPE A IN THE TREATMENT OF SPASTICITY IN CHILDREN WITH CEREBRAL PALSY

    Directory of Open Access Journals (Sweden)

    Ljiljana Lazić

    2011-06-01

    Full Text Available Cerebral palsy has an incidence of about 1-2 per 1000 live births, and in spite of the progress of neonatal medicine, it seems that the incidence will not subside in the near future. The most important characteristic of cerebral palsy is movement abnormality: spasticity, chorea, athetosis, ataxia, dystonia, as well as their different combinations. About 70% of children who suffer from cerebral palsy also suffer from some form of spasticity. Spasticity is a type of muscle hypertonicity characterized by rapid increase in resistance to passive stretching of muscles. The interest for botulinum toxin application in the treatment of spasticity has dramatically increased in the last 10 years. Botulinum toxin is the most powerful neurotoxin that is found in nature. It is produced by anaerobic bacteria – clostridium botulinum. It is produced in eight serotypes of which type A is the most commonly used. Botulinum toxin blocks neuromuscular transmission and causes irreversible weakness of the treated muscle. It has been used since 1993 in the treatment of cerebral palsy in children. The toxin effect is permanent and it results in irreversible denervation. Functional recovery is possible after 2-4 months, due to sprouting of nerve endings and the formation of new synaptic contacts. Treatment with botulinum toxin is safe. Adverse effects are rare, temporary and completely reversible. Application of botulinum toxin prevents or reduces contractures and deformities, and thus delays or avoids surgical treatment. Yet, physical therapy, which prolongs and improves the effects of botulinum toxin, remains an essential and most important form of therapy in the treatment of children with cerebral palsy.

  7. Rapid Generation of a Nanocrystal-Labeled Peptide Library for Specific Identification of the Bacterium Clostrium Botulinum

    Energy Technology Data Exchange (ETDEWEB)

    Tok, J B

    2004-11-11

    Several peptide libraries containing up to 2 million unique peptide ligands have been synthesized. The peptides are attached onto a 80 micron resin and the length of these peptide ligands ranges from 5 to 9 amino acid residues. Using a novel calorimetric assay, the libraries were screened for binding to the ganglioside-binding domain of Clostridium Tetanus Toxin, a structural similar analog of the Clostridium Botulinum toxin. Several binding peptide sequences were identified, in which the detailed binding kinetics are currently underway using the Surface Plasmon Resonance (SPR) technique.

  8. Rapid Generation of a Nanocrystal-Labeled Peptide Library for Specific Identification of the Bacterium Clostrium Botulinum

    International Nuclear Information System (INIS)

    Tok, J B

    2004-01-01

    Several peptide libraries containing up to 2 million unique peptide ligands have been synthesized. The peptides are attached onto a 80 micron resin and the length of these peptide ligands ranges from 5 to 9 amino acid residues. Using a novel calorimetric assay, the libraries were screened for binding to the ganglioside-binding domain of Clostridium Tetanus Toxin, a structural similar analog of the Clostridium Botulinum toxin. Several binding peptide sequences were identified, in which the detailed binding kinetics are currently underway using the Surface Plasmon Resonance (SPR) technique

  9. Novel complex formed between a nonproteolytic cell wall protein of group A streptococci and α2-macroglobulin

    International Nuclear Information System (INIS)

    Chhatwal, G.S.; Albohn, G.; Blobel, H.

    1987-01-01

    Binding of 125 I-labeled α 2 -macroglobulin (α 2 M) to streptococci belonging to serological groups A, B, C, and G was studied. Streptococci of groups A and G interacted only with native α 2 M, and those of group C reacted only with α 2 M-trypsin complex. Binding of α 2 M to group A streptococci was saturable and reversible. The dissociation constant was 2.02 x 10 -7 M, and the number of binding sites was calculated to be 18,000 per streptococcus. The α 2 M-binding protein could be solubilized by treatment of group A streptococci with a murolytic enzyme and subsequently purified by affinity chromatography and high-pressure liquid chromatography. The purified protein was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 78,000. It possessed no proteolytic activity and interacted with native α 2 M in Western blots (immunoblots). Interaction of purified binding protein with α 2 M led to a change in the conformation of α 2 M similar to that obtained by α 2 M-protease complexes. Reversible binding of a nonproteolytic streptococcal component of α 2 M is thus a novel feature of α 2 M reactivity

  10. Botulinum Toxin for Neuropathic Pain: A Review of the Literature

    Directory of Open Access Journals (Sweden)

    Hyun-Mi Oh

    2015-08-01

    Full Text Available Botulinum neurotoxin (BoNT, derived from Clostridium botulinum, has been used therapeutically for focal dystonia, spasticity, and chronic migraine. Its spectrum as a potential treatment for neuropathic pain has grown. Recent opinions on the mechanism behind the antinociceptive effects of BoNT suggest that it inhibits the release of peripheral neurotransmitters and inflammatory mediators from sensory nerves. There is some evidence showing the axonal transport of BoNT, but it remains controversial. The aim of this review is to summarize the experimental and clinical evidence of the antinociceptive effects, mechanisms, and therapeutic applications of BoNT for neuropathic pain conditions, including postherpetic neuralgia, complex regional pain syndrome, and trigeminal neuralgia. The PubMed and OvidSP databases were searched from 1966 to May 2015. We assessed levels of evidence according to the American Academy of Neurology guidelines. Recent studies have suggested that BoNT injection is an effective treatment for postherpetic neuralgia and is likely efficient for trigeminal neuralgia and post-traumatic neuralgia. BoNT could also be effective as a treatment for diabetic neuropathy. It has not been proven to be an effective treatment for occipital neuralgia or complex regional pain syndrome.

  11. Botulinum Toxin A for Bladder Pain Syndrome/Interstitial Cystitis

    Directory of Open Access Journals (Sweden)

    Bin Chiu

    2016-07-01

    Full Text Available Botulinum neurotoxin A (BoNT-A, derived from Clostridium botulinum, has been used clinically for several diseases or syndrome including chronic migraine, spasticity, focal dystonia and other neuropathic pain. Chronic pelvic or bladder pain is the one of the core symptoms of bladder pain syndrome/interstitial cystitis (BPS/IC. However, in the field of urology, chronic bladder or pelvic pain is often difficult to eradicate by oral medications or bladder instillation therapy. We are looking for new treatment modality to improve bladder pain or associated urinary symptoms such as frequency and urgency for patients with BPS/IC. Recent studies investigating the mechanism of the antinociceptive effects of BoNT A suggest that it can inhibit the release of peripheral neurotransmitters and inflammatory mediators from sensory nerves. In this review, we will examine the evidence supporting the use of BoNTs in bladder pain from basic science models and review the clinical studies on therapeutic applications of BoNT for BPS/IC.

  12. Botulinum Toxin A for Bladder Pain Syndrome/Interstitial Cystitis.

    Science.gov (United States)

    Chiu, Bin; Tai, Huai-Ching; Chung, Shiu-Dong; Birder, Lori A

    2016-07-01

    Botulinum neurotoxin A (BoNT-A), derived from Clostridium botulinum, has been used clinically for several diseases or syndrome including chronic migraine, spasticity, focal dystonia and other neuropathic pain. Chronic pelvic or bladder pain is the one of the core symptoms of bladder pain syndrome/interstitial cystitis (BPS/IC). However, in the field of urology, chronic bladder or pelvic pain is often difficult to eradicate by oral medications or bladder instillation therapy. We are looking for new treatment modality to improve bladder pain or associated urinary symptoms such as frequency and urgency for patients with BPS/IC. Recent studies investigating the mechanism of the antinociceptive effects of BoNT A suggest that it can inhibit the release of peripheral neurotransmitters and inflammatory mediators from sensory nerves. In this review, we will examine the evidence supporting the use of BoNTs in bladder pain from basic science models and review the clinical studies on therapeutic applications of BoNT for BPS/IC.

  13. Botulinum toxin (Botox) to enhance facial macroesthetics: a literature review.

    Science.gov (United States)

    Dastoor, Sarosh F; Misch, Carl E; Wang, Hom-Lay

    2007-01-01

    Dental implants have emerged as a predictable treatment option for partial edentulism. Their ability to preserve bone and soft tissue yields highly esthetic results in the long term. Increasingly, patients are demanding not only enhancements to their dental (micro) esthetics but also to their overall facial (macro) esthetics. Dynamic wrinkles (caused by hyperfunctional muscles) in the perioral, glabellar, and forehead regions can cause a patient's expressions to be misinterpreted as angry, anxious, fearful, or fatigued. An emerging treatment option to address these issues is the use of a paralyzing material such as botulinum toxin A (Botox) to decrease the appearance of the wrinkles, which yields a more esthetic and youthful facial appearance. Botox is a deadly poison that is produced by the bacterium Clostridium botulinum and causes muscle paralysis by inhibiting acetylcholine release at the neuromuscular junction. When used in areas of hyperfunctional muscles, a transient partial paralysis occurs that diminishes the appearances of wrinkles, Therefore, wrinkles not attributable to hyperfunctional muscles (e.g., wrinkles caused by aging, gravity, photodamage, trauma, and scarring) will not be amenable to treatment with the toxin. As a result, proper case selection is essential. A thorough understanding of the indications, techniques, dosages, and complications and their management is imperative to achieve a satisfactory result. This article will review the pathogenesis of facial wrinkles as well as the history, techniques, clinical controversies, and other important considerations for successful treatment of facial wrinkles with Botox.

  14. Genetic diversity within the botulinum neurotoxin-producing bacteria and their neurotoxins.

    Science.gov (United States)

    Hill, K K; Xie, G; Foley, B T; Smith, T J

    2015-12-01

    The recent availability of multiple Clostridium botulinum genomic sequences has initiated a new genomics era that strengthens our understanding of the bacterial species that produce botulinum neurotoxins (BoNTs). Analysis of the genomes has reinforced the historical Group I-VI designations and provided evidence that the bont genes can be located within the chromosome, phage or plasmids. The sequences provide the opportunity to examine closely the variation among the toxin genes, the composition and organization of the toxin complex, the regions flanking the toxin complex and the location of the toxin within different bacterial strains. These comparisons provide evidence of horizontal gene transfer and site-specific insertion and recombination events that have contributed to the variation observed among the neurotoxins. Here, examples that have contributed to the variation observed in serotypes A-H strains are presented to illustrate the mechanisms that have contributed to their variation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Investigations into Small Molecule Non-Peptidic Inhibitors of the Botulinum Neurotoxins

    Science.gov (United States)

    Čapková, Kateřina; Salzameda, Nicholas T.; Janda, Kim D.

    2009-01-01

    Botulinum neurotoxins (BoNTs), proteins secreted by the bacteria genus Clostridium, represent a group of extremely lethal toxins and a potential bioterrorism threat. As the current therapeutic options are of a predominantly prophylactic nature and cannot be used en masse, new strategies and ultimately potential treatments are desperately needed to combat any widespread release of these neurotoxins. In these regards, our laboratory has been working on developing new alternatives to treat botulinum intoxication through the development of inhibitors of the light chain proteases, the etiological agent which causes BoNT intoxication. Such a strategy has required the construction of two high throughput screens and small molecule non-peptidic libraries; excitingly, inhibitors of the BoNT/A protease have been uncovered and are being optimized via structure activity relationship studies. PMID:19327377

  16. Collagenase Clostridium Histolyticum Injection

    Science.gov (United States)

    ... disease (a thickening of tissue [plaque] inside the penis that causes the penis to curve). Collagenase Clostridium histolyticum injection is in ... the plaque of thickened tissue and allows the penis to be straightened.

  17. Clostridium XIV Meeting

    Energy Technology Data Exchange (ETDEWEB)

    Lynd, Lee

    2016-08-28

    The 14th biannual Clostridium meeting was held at Dartmouth College from August 28 through 31, 2016. As noted in the meeting program (http://clostridiumxiv.com/wp-content/uploads/2016/09/Clostridium_XIV_program.pdf). the meeting featured 119 registered attendees, 33 oral presentations, 5 of which were given by younger presenters, 40 posters, and 2 keynote presentations, with strong participation by female and international scientists.

  18. Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples▿

    Science.gov (United States)

    Kirchner, Sebastian; Krämer, K. Melanie; Schulze, Martin; Pauly, Diana; Jacob, Daniela; Gessler, Frank; Nitsche, Andreas; Dorner, Brigitte G.; Dorner, Martin B.

    2010-01-01

    Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 103 and 105 CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F. PMID:20435756

  19. CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C.

    Directory of Open Access Journals (Sweden)

    Kentaro Tsukamoto

    Full Text Available Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9 system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

  20. PCR multiplex para identificação de isolados de Clostridium chauvoei e Clostridium septicum Multiplex PCR for identification of Clostridium chauvoei and Clostridium septicum

    Directory of Open Access Journals (Sweden)

    R.A. Assis

    2008-04-01

    Full Text Available Padronizou-se uma técnica de reação em cadeia da polimerase múltipla (PCR multiplex para detecção de Clostridium chauvoei e Clostridium septicum em culturas puras. Foram utilizados pares de iniciadores para segmentos específicos dos genes que codificam a flagelina de C. chauvoei e a toxina alfa de C. septicum. Para avaliaçã o da PCR multiplex, foram testados 16 isolados clínicos de C. chauvoei e 15 isolados de C. septicum provenientes de ruminantes, quatro sementes vacinais de cada um desses agentes. Amostras de referência de ambos os microrganismos foram usadas como controle. Para avaliar a especificidade, DNAs genômicos dos seguintes microrganismos foram usados: C. sordellii, C. novyi tipo A, C. novyi tipo B, C. perfringens tipo A, C. haemolyticum, C. botulinum tipo D, Pseudomonas aeruginosa, Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli e Salmonella typhimurium. Todos os isolados e sementes vacinais de C. chauvoei e C. septicum foram detectados pela técnica. Não foram observadas reações cruzadas com as outras espécies de clostrídios, outras espécies bacterianas ou entre C. Chauvoei e C. septicum. As menores concentrações de DNA de C. chauvoei e C. septicum detectadas foram 45pg/µl e 30pg/µl, respectivamente. A PCR multiplex pode ser utilizada para a identificação específica de C. chauvoei e C. septicum em culturas puras.Multiplex PCR was optimized to detect Clostridium chauvoei and Clostridium septicum in pure cultures. In each reaction, a pair of primers for a specific segment of the flagellin gene of C. chauvoei and a pair of primers for a specific segment of alpha toxin gene of C. septicum were employed. Reference strains of both microorganisms were used as control. The multiplex PCR was evaluated by testing 16 clinical isolates of C. chauvoei from ruminants, 15 clinical isolates of C. septicum from ruminants and, four vaccine strains of each one of these agents. Reference strains of both

  1. Prevalence of C. botulinum and C. perfringens spores in food products available on Polish market

    Directory of Open Access Journals (Sweden)

    Grenda Tomasz

    2017-09-01

    Full Text Available Introduction: The aim of this study was to evaluate the prevalence of Clostridium botulinum and Clostridium perfringens in food samples purchased from Polish producers. Material and Methods: The analyses were performed on 260 food samples collected in Lublin and Subcarpathian regions: 56 of smoked meat, 21 of pork meat, 20 of dairy products, 26 of vegetable and fruit preserves, 40 of ready-to-eat meals, 27 of fish preserves, and 70 of honey collected directly from apiaries. Results: C. botulinum strains were isolated from 2.3% (6/260 of samples and the isolates were classified as toxin types A (4/260 and B (2/260. C. perfringens strains were isolated from 14% (37/260 of samples. All the isolates were classified as toxin type A, 28 of them were able also to produce α toxin and 9 - β2 toxin. Conclusion: On the basis of the obtained results it could be suggested that risk assessment, especially regarding the entire honey harvesting process, should be provided in order to ensure the microbiological safety of the products to be consumed by infants and people with a weakened immune system.

  2. Botulinum neurotoxins: mechanism of action.

    Science.gov (United States)

    Tighe, Ann P; Schiavo, Giampietro

    2013-06-01

    Botulinum neurotoxins are used clinically for conditions characterized by hyperexcitability of peripheral nerve terminals and hypersecretory syndromes. These neurotoxins are synthesized as precursor proteins with low activity, but their effects are mediated by the active form of the neurotoxin through a multistep mechanism. Following a high-affinity interaction with a protein receptor and polysialogangliosides on the synaptic membrane, botulinum neurotoxins enter the neuron and causes a sustained inhibition of synaptic transmission. The active neurotoxin is part of a high-molecular-weight complex that protects the neurotoxin from proteolytic degradation. Although complexing proteins do not affect diffusion of therapeutic neurotoxin, they may lead to the development of neutralizing antibodies that block responsiveness to it. Nerve terminal intoxication is reversible and its duration varies for different BoNT serotypes. Although it was previously assumed that botulinum neurotoxins exert effects only on the peripheral synapses, such as the neuromuscular junction, there is now substantial evidence that these neurotoxins affect neurotransmission at distal central nervous system sites as well. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Botulinum toxin — therapeutic effect in cosmetology

    OpenAIRE

    Morrison A.V.; Bocharova Y.M.; Morrison V.V.

    2016-01-01

    This review presents the data from published literatures and the research works conducted by the authors about mechanisms of action of botulinum toxin and its use in the practical medicine (particularly in dermatology and cosmetology). Indications and contraindications of botulinum toxin use in cosmetology are also considered in this work.

  4. Building-block architecture of botulinum toxin complex: Conformational changes provide insights into the hemagglutination ability of the complex

    Directory of Open Access Journals (Sweden)

    Tomonori Suzuki

    2017-03-01

    Full Text Available Clostridium botulinum produces the botulinum neurotoxin (BoNT. Previously, we provided evidence for the “building-block” model of botulinum toxin complex (TC. In this model, a single BoNT is associated with a single nontoxic nonhemagglutinin (NTNHA, yielding M-TC; three HA-70 molecules are attached and form M-TC/HA-70, and one to three “arms” of the HA-33/HA-17 trimer (two HA-33 and one HA-17 further bind to M-TC/HA-70 via HA-17 and HA-70 binding, yielding one-, two-, and three-arm L-TC. Of all TCs, only the three-arm L-TC caused hemagglutination. In this study, we determined the solution structures for the botulinum TCs using small-angle X-ray scattering (SAXS. The mature three-arm L-TC exhibited the shape of a “bird spreading its wings”, in contrast to the model having three “arms”, as revealed by transmission electron microscopy. SAXS images indicated that one of the three arms of the HA-33/HA-17 trimer bound to both HA-70 and BoNT. Taken together, these findings regarding the conformational changes in the building-block architecture of TC may explain why only three-arm L-TC exhibited hemagglutination.

  5. Optimization of peptide substrates for botulinum neurotoxin E improves detection sensitivity in the Endopep-MS assay.

    Science.gov (United States)

    Wang, Dongxia; Krilich, Joan; Baudys, Jakub; Barr, John R; Kalb, Suzanne R

    2015-01-01

    Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances known to humankind. It is essential to have a simple, quick, and sensitive method for the detection and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory has developed a mass spectrometry-based Endopep-MS assay that is able to rapidly detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of seven distinctive BoNT serotypes (A-G) and is the causative agent of botulism in both humans and animals. To improve the sensitivity of the Endopep-MS assay, we report here the development of novel peptide substrates for the detection of BoNT/E activity through systematic and comprehensive approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement in sensitivity compared with the current substrate for the detection of both not-trypsin-activated and trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouse LD50/ml was achieved using the novel peptide substrate in the assay to detect not-trypsin-activated BoNT/E complex spiked in serum, stool, and food samples. Published by Elsevier Inc.

  6. Clostridium difficile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria.

    Directory of Open Access Journals (Sweden)

    Carsten Schwan

    2009-10-01

    Full Text Available Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by production of the Rho GTPase-glucosylating toxins A and B. Recently emerging hypervirulent Clostridium difficile strains additionally produce the binary ADP-ribosyltransferase toxin CDT (Clostridium difficile transferase, which ADP-ribosylates actin and inhibits actin polymerization. Thus far, the role of CDT as a virulence factor is not understood. Here we report by using time-lapse- and immunofluorescence microscopy that CDT and other binary actin-ADP-ribosylating toxins, including Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin, induce redistribution of microtubules and formation of long (up to >150 microm microtubule-based protrusions at the surface of intestinal epithelial cells. The toxins increase the length of decoration of microtubule plus-ends by EB1/3, CLIP-170 and CLIP-115 proteins and cause redistribution of the capture proteins CLASP2 and ACF7 from microtubules at the cell cortex into the cell interior. The CDT-induced microtubule protrusions form a dense meshwork at the cell surface, which wrap and embed bacterial cells, thereby largely increasing the adherence of Clostridia. The study describes a novel type of microtubule structure caused by less efficient microtubule capture and offers a new perspective for the pathogenetic role of CDT and other binary actin-ADP-ribosylating toxins in host-pathogen interactions.

  7. An Investigation of Immunogenicity of Chitosan-Based Botulinum Neurotoxin E Binding Domain Recombinant Candidate Vaccine via Mucosal Route

    Directory of Open Access Journals (Sweden)

    Mohammad Javad Bagheripour

    2017-01-01

    Full Text Available Background and Objectives: Botulism syndrome is caused by serotypes A-G of neurotoxins of Clostridium genus. Neurotoxin binding domain is an appropriate vaccine candidate due to its immunogenic activity. In this study, the immunogenicity of chitosan-based botulinum neurotoxin E binding domain recombinant candidate vaccine was investigated via mucosal route of administration. Methods: In this experimental study, chitosan nanoparticles containing rBoNT/E protein were synthesized by ionic gelation method and were administered orally and intranasally to mice. After each administration, IgG antibody titer was measured by ELISA method. Finally, all groups were challenged with active botulinum neurotoxin type E. Data were analyzed using Duncan and repeated ANOVA tests. The significance level was considered as p0.05, even intranasal route reduced the immunogenicity.

  8. Regulation of toxin and bacteriocin synthesis in Clostridium species by a new subgroup of RNA polymerase sigma-factors.

    Science.gov (United States)

    Dupuy, Bruno; Matamouros, Susana

    2006-04-01

    Many Clostridium species are pathogenic for humans and animals, and most of the resulting diseases, such as tetanus, botulism, gas gangrene and pseudomembranous colitis, are due to the production of potent extracellular toxins. The biochemical mechanisms of action of Clostridium toxins have been extensively studied in the past ten years. However, detailed information about the regulation of toxin gene expression has only recently emerged. TcdR, BotR, TetR and UviA are now known to be related alternative RNA polymerase sigma factors that drive transcription of toxin A and toxin B genes in C. difficile, the neurotoxin genes in C. botulinum and C. tetani, and a bacteriocin gene in C. perfringens. Although the Clostridium sigma factors have some similarity to members of the ECF sigma factor group, they differ sufficiently in structure and function so that they have been assigned to a new group within the sigma(70)-family.

  9. Clostridium difficile Infection

    Science.gov (United States)

    ... TeensRead MoreBMI Calculator Acute BronchitisHigh Blood PressureBursitis of the HipHigh CholesterolExercise-induced UrticariaMicroscopic HematuriaKidney CystsDe Quervain’s Tenosynovitis Home Diseases and Conditions Clostridium difficile (C. diff.) ...

  10. Bacteriophages of Clostridium perfringens

    Science.gov (United States)

    The specific aims of the book chapter are to: (1) Briefly review the nomenclature of bacteriophages and how these agents are classified. (2) Discuss the problems associated with addition/removal of antibiotics in commercial animal feeds. (3) Provide a brief overview of Clostridium perfringens biolog...

  11. Clostridium tetani bacteraemia.

    Science.gov (United States)

    Hallit, Rabih Riad; Afridi, Muhammad; Sison, Raymund; Salem, Elie; Boghossian, Jack; Slim, Jihad

    2013-01-01

    Tetanus is a neuromuscular disease in which Clostridium tetani exotoxin (tetanospasmin) produces muscle spasms, incapacitating its host. To our knowledge, C. tetani bacteraemia has never been reported in the literature. The ideal management of this entity remains unresolved given that there is no literature to guide the therapy.

  12. Evaluation of CP Chromo Select Agar for the enumeration of Clostridium perfringens from water.

    Science.gov (United States)

    Manafi, Mammad; Waldherr, Kerstin; Kundi, Michael

    2013-10-01

    The European Directive on drinking water quality has included mCP agar as the reference method for recovering Clostridium perfringens from drinking waters. In the present study, three media (mCP, TSCF and CP Chromo Select Agar) were evaluated for recovery of C. perfringens in different surface water samples. Out of 139 water samples, using a membrane filtration technique, 131 samples (94.2%) were found to be presumptively positive for C. perfringens in at least one of the culture media. Green colored colonies on CP Chromo Select Agar (CCP agar) were counted as presumptive C. perfringens isolates. Out of 483 green colonies on CCP agar, 96.3% (465 strains, indole negative) were identified as C. perfringens, and 15 strains (3.1%) were indole positive and were identified as Clostridium sordellii, Clostridium bifermentans or Clostridium tetani. Only 3 strains (0.6%) gave false positive results and were identified as Clostridium fallax, Clostridium botulinum, and Clostridium tertium. Variance analysis of the data obtained shows statistically no significant differences in the counts obtained between media employed in this work. The mCP method is very onerous for routine screening and bacterial colonies could not be used for further biochemical testing. The colonies on CCP and TSCF were easy to count and subculture for confirmation tests. TSCF detects sulfite-reducing clostridia, including species other than C. perfringens, and in some cases excessive blackening of the agar frustrated counting of the colonies. If the contamination was too high, TSCF did not consistently produce black colonies and as a consequence, the colonies were white and gave false negative results. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that CCP agar was the most useful medium for C. perfringens recovery in water samples. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Taxonogenomic description of four new Clostridium species isolated from human gut: ‘Clostridium amazonitimonense’, ‘Clostridium merdae’, ‘Clostridium massilidielmoense’ and ‘Clostridium nigeriense’

    Directory of Open Access Journals (Sweden)

    M.T. Alou

    2018-01-01

    Full Text Available Culturomics investigates microbial diversity of the human microbiome by combining diversified culture conditions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rRNA gene identification. The present study allowed identification of four putative new Clostridium sensu stricto species: ‘Clostridium amazonitimonense’ strain LF2T, ‘Clostridium massilidielmoense’ strain MT26T, ‘Clostridium nigeriense’ strain Marseille-P2414T and ‘Clostridium merdae’ strain Marseille-P2953T, which we describe using the concept of taxonogenomics. We describe the main characteristics of each bacterium and present their complete genome sequence and annotation.

  14. TetR Is a Positive Regulator of the Tetanus Toxin Gene in Clostridium tetani and Is Homologous to BotR

    NARCIS (Netherlands)

    Marvaud, Jean-Christophe; Eisel, Ulrich; Binz, Thomas; Niemann, Heiner; Popoff, Michel R.

    1998-01-01

    The TetR gene immediately upstream from the tetanus toxin (TeTx) gene was characterized. It encodes a 21,562-Da protein which is related (50 to 65% identity) to the equivalent genes (botR) in Clostridium botulinum. TetR has the feature of a DNA binding protein with a basic pI (9.53). It contains a

  15. Botulinum A toxin utilizations in obstetric palsy

    Directory of Open Access Journals (Sweden)

    Atakan Aydin

    2012-12-01

    Conclusion: We conclude that with the help of botulinum A toxin and physyotherapy, obstetrical palsy patient with cocontractions can significantly improve movements and may have less surgery. [Hand Microsurg 2012; 1(3.000: 89-94

  16. Botulinum toxin for the treatment of strabismus.

    Science.gov (United States)

    Rowe, Fiona J; Noonan, Carmel P

    2017-03-02

    The use of botulinum toxin as an investigative and treatment modality for strabismus is well reported in the medical literature. However, it is unclear how effective it is in comparison to other treatment options for strabismus. The primary objective was to examine the efficacy of botulinum toxin therapy in the treatment of strabismus compared with alternative conservative or surgical treatment options. This review sought to ascertain those types of strabismus that particularly benefit from the use of botulinum toxin as a treatment option (such as small angle strabismus or strabismus with binocular potential, i.e. the potential to use both eyes together as a pair). The secondary objectives were to investigate the dose effect and complication rates associated with botulinum toxin. We searched CENTRAL (which contains the Cochrane Eyes and Vision Trials Register) (2016, Issue 6), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to July 2016), Embase (January 1980 to July 2016), Latin American and Caribbean Literature on Health Sciences (LILACS) (January 1982 to July 2016), the ISRCTN registry (www.isrctn.com/editAdvancedSearch), ClinicalTrials.gov (www.clinicaltrials.gov), and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 11 July 2016. We handsearched the British and Irish Orthoptic Journal, Australian Orthoptic Journal, proceedings of the European Strabismological Association (ESA), International Strabismological Association (ISA) and International Orthoptic Association (IOA) (www.liv.ac.uk/orthoptics/research/search.htm) and American Academy of Paediatric Ophthalmology and Strabismus meetings (AAPOS). We contacted researchers who are active in this field for information about further

  17. Botulinum toxin for the treatment of bruxism.

    Science.gov (United States)

    Tinastepe, Neslihan; Küçük, Burcu Bal; Oral, Koray

    2015-10-01

    Botulinum toxin, the most potent biological toxin, has been shown to be effective for a variety of disorders in several medical conditions, when used both therapeutically and cosmetically. In recent years, there has been a rising trend in the use of this pharmacological agent to control bruxing activity, despite its reported adverse effects. The aim of this review was to provide a brief overview to clarify the underlying essential ideas for the use of botulinum toxin in bruxism based on available scientific papers. An electronic literature search was performed to identify publications related to botulinum toxin and its use for bruxism in PubMed. Hand searching of relevant articles was also made to identify additional studies. Of the eleven identified studies, only two were randomized controlled trials, compared with the effectiveness of botulinum toxins on the reduction in the frequency of bruxism events and myofascial pain after injection. The authors of these studies concluded that botulinum toxin could be used as an effective treatment for reducing nocturnal bruxism and myofascial pain in patients with bruxism. Evidence-based research was limited on this topic. More randomized controlled studies are needed to confirm that botulinum toxin is safe and reliable for routine clinical use in bruxism.

  18. Characterization of Clostridium Species from Food Commodities and Faecal Specimens in Lagos State, Nigeria.

    Science.gov (United States)

    Chukwu, E E; Ogunsola, F T; Nwaokorie, F O; Coker, A O

    2015-01-01

    Food-borne pathogens are a major public health challenge worldwide. These organisms' cause illnesses leading to time loss in the work place and reduced productivity.Clostridium species cause infections through the production of powerful toxins which are responsible for diarrhoea and cramping. Diarrhoeal diseases due to Clostridia are one of the commonest worldwide but have hardly been reported in Nigeria. This study characterized Clostridium species from food commodities and human faeces in Lagos State MATERIALS AND METHODS: Four hundred and twenty samples comprising food (220) and faecal (200) specimens in Lagos state were included in this study. Isolates obtained were identified using API 20-A and confirmed by Polymerase Chain Reaction assay and 16S rRNA sequencing. The food samples included meat and meat products, ready to drink traditionally prepared milk products, fresh vegetables, canned foods and local honey. Seventy (16.7%) Clostridium species were identified, 50 from food and 20 from faeces. Majority of the isolates were obtained from vegetables (56%) and meat products (34%). Of the 70 Clostridial species, 38 (54.3%) were C. perfringens, 5 (7.1%) were C. difficile and 2 (2.9%) were C. botulinum. All 38 (100%) strains of C. perfringens possessed alpha (cpa) toxin gene. Clostridium species are present in our environment and contaminate food products posing potential risks to consumers. There is therefore a need for these traditionally made street vended foods to be monitored because they are potential sources of food borne pathogens.

  19. Rational Design of Therapeutic and Diagnostic Against Botulinum Neurotoxin

    National Research Council Canada - National Science Library

    Chan, N. W; Wang, Y; Tenn, C. C; Weiss, Ten; Hancock, J. R; Chenler, C. L; Lee, W. E; Dickinson-Laing, T; Yin, J; Gebremedhin, M. G; Mah, D. C

    2006-01-01

    .... In spite of botulinum neurotoxin being the most poisonous material, little is known about its mechanism of binding, effective drugs are lacking, and correct diagnosis of botulinum poisoning is slow...

  20. Peptide inhibitors of botulinum neurotoxin serotype A: design, inhibition, cocrystal structures, structure-activity relationship and pharmacophore modeling

    Energy Technology Data Exchange (ETDEWEB)

    Kumar G.; Swaminathan S.; Kumaran, D.; Ahmed, S. A.

    2012-05-01

    Clostridium botulinum neurotoxins are classified as Category A bioterrorism agents by the Centers for Disease Control and Prevention (CDC). The seven serotypes (A-G) of the botulinum neurotoxin, the causative agent of the disease botulism, block neurotransmitter release by specifically cleaving one of the three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and induce flaccid paralysis. Using a structure-based drug-design approach, a number of peptide inhibitors were designed and their inhibitory activity against botulinum serotype A (BoNT/A) protease was determined. The most potent peptide, RRGF, inhibited BoNT/A protease with an IC{sub 50} of 0.9 {micro}M and a K{sub i} of 358 nM. High-resolution crystal structures of various peptide inhibitors in complex with the BoNT/A protease domain were also determined. Based on the inhibitory activities and the atomic interactions deduced from the cocrystal structures, the structure-activity relationship was analyzed and a pharmacophore model was developed. Unlike the currently available models, this pharmacophore model is based on a number of enzyme-inhibitor peptide cocrystal structures and improved the existing models significantly, incorporating new features.

  1. Toxina botulínica y su empleo en la patología oral y maxilofacial Botulinum toxin and its use in oral and maxillofacial pathology

    Directory of Open Access Journals (Sweden)

    D. Martínez-Pérez

    2004-06-01

    Full Text Available Resumen: Las toxinas botulínicas son exotoxinas de la bacteria formadora de esporas Clostridim botulinum y los agentes causantes del botulismo. Cuando se inyecta en el músculo produce una parálisis flácida. El efecto clínico está directamente relacionado con la dosis y debe ajustarse para cada caso concreto. La Toxina botulínica ha demostrado en los más de veinte años en que se está utilizando que es un fármaco seguro. Las indicaciones de la toxina botulínica en la actualidad incluyen todas aquellas patologías que resultan de la hiperfunción muscular y la disfunción autonómica.Abstract: Botilinum toxins are exotoxins of the bacteria that form the Clostridium botulinum spores and the causative agents of botulism. When injected into the muscle flaccid paralysis is produced. The clinical effect is directly related with the dose and is should be adjusted for each particular case. over the last twenty years that it has been in use, the botulinum toxin has shown itself to be a reliable drug. Current indications for the use of botulinum toxin include all those pathologies which are the results of muscle hyperfunction and autonomic dysfunction.

  2. A Monoclonal Antibody Based Capture ELISA for Botulinum Neurotoxin Serotype B: Toxin Detection in Food

    Directory of Open Access Journals (Sweden)

    Larry H. Stanker

    2013-11-01

    Full Text Available Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT, produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A–H have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD’s for individual antibodies ranging from 10 to 48 × 10−11 M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D., ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule and readily detects toxin in those food samples tested.

  3. The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies

    Directory of Open Access Journals (Sweden)

    Christine Rasetti-Escargueil

    2017-10-01

    Full Text Available The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies. For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs. The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high “humanness” predicts a high tolerance in humans.

  4. Substrate Binding Mode and its Implication on Drug Design for Botulinum Neurotoxin A

    Energy Technology Data Exchange (ETDEWEB)

    Kumaran, D.; Rawat, R; Ahmed, A; Swaminathan, S

    2008-01-01

    The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins, the causative agents of botulism, block the neurotransmitter release by specifically cleaving one of the three SNARE proteins and induce flaccid paralysis. The Centers for Disease Control and Prevention (CDC) has declared them as Category A biowarfare agents. The most potent among them, botulinum neurotoxin type A (BoNT/A), cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25). An efficient drug for botulism can be developed only with the knowledge of interactions between the substrate and enzyme at the active site. Here, we report the crystal structures of the catalytic domain of BoNT/A with its uncleavable SNAP-25 peptide 197QRATKM202 and its variant 197RRATKM202 to 1.5 A and 1.6 A, respectively. This is the first time the structure of an uncleavable substrate bound to an active botulinum neurotoxin is reported and it has helped in unequivocally defining S1 to S5? sites. These substrate peptides make interactions with the enzyme predominantly by the residues from 160, 200, 250 and 370 loops. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197) chelate the zinc ion and replace the nucleophilic water. The P1?-Arg198, occupies the S1? site formed by Arg363, Thr220, Asp370, Thr215, Ile161, Phe163 and Phe194. The S2? subsite is formed by Arg363, Asn368 and Asp370, while S3? subsite is formed by Tyr251, Leu256, Val258, Tyr366, Phe369 and Asn388. P4?-Lys201 makes hydrogen bond with Gln162. P5?-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop. Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin.

  5. Substrate binding mode and its implication on drug design for botulinum neurotoxin A.

    Directory of Open Access Journals (Sweden)

    Desigan Kumaran

    2008-09-01

    Full Text Available The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins, the causative agents of botulism, block the neurotransmitter release by specifically cleaving one of the three SNARE proteins and induce flaccid paralysis. The Centers for Disease Control and Prevention (CDC has declared them as Category A biowarfare agents. The most potent among them, botulinum neurotoxin type A (BoNT/A, cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25. An efficient drug for botulism can be developed only with the knowledge of interactions between the substrate and enzyme at the active site. Here, we report the crystal structures of the catalytic domain of BoNT/A with its uncleavable SNAP-25 peptide (197QRATKM(202 and its variant (197RRATKM(202 to 1.5 A and 1.6 A, respectively. This is the first time the structure of an uncleavable substrate bound to an active botulinum neurotoxin is reported and it has helped in unequivocally defining S1 to S5' sites. These substrate peptides make interactions with the enzyme predominantly by the residues from 160, 200, 250 and 370 loops. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197 chelate the zinc ion and replace the nucleophilic water. The P1'-Arg198, occupies the S1' site formed by Arg363, Thr220, Asp370, Thr215, Ile161, Phe163 and Phe194. The S2' subsite is formed by Arg363, Asn368 and Asp370, while S3' subsite is formed by Tyr251, Leu256, Val258, Tyr366, Phe369 and Asn388. P4'-Lys201 makes hydrogen bond with Gln162. P5'-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop. Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin.

  6. Pré-compostagem de cadáveres de bovinos acometidos pelo botulismo The effect of composting cattle carcasses on botulinum spores and toxin content

    Directory of Open Access Journals (Sweden)

    Vera C.L.M. Curci

    2007-04-01

    Full Text Available Foram pré-compostados, em células individuais e isoladas, cinco cadáveres de bovinos acometidos pelo botulismo com a finalidade de monitorar a presença de esporos de Clostridium botulinum e de toxina botulínica antes e após o processo de decomposição em leira estática com fonte de carbono. O diagnóstico da intoxicação nos animais foi baseado nas características clínico-patológicas, epidemiológicas ou laboratoriais. Dos cinco bovinos com evolução clínica crônica de botulismo, cujos cadáveres foram pré-compostados, três foram acometidos pela toxina tipo D, um pelo complexo CD e um dos animais foi negativo na tentativa de detecção da toxina e de esporos da bactéria nas vísceras pelo bioensaio e neutralização em camundongo. O processo de pré-compostagem foi realizado em leira estática, com o uso de material carbonáceo umidificado como substrato e esquartejamento do animal, vedado individualmente com lona plástica e sem aeração por um período de 50 dias. A temperatura das leiras foi monitorada durante o período e oscilou de 40,5-52,4ºC. Após a abertura das leiras, pôde-se constatar a completa decomposição de todo material mole, com redução significativa do seu peso (de 26,5-44,5%, restando apenas os ossos. Não foi detectado esporo ou toxina botulínica no interior dos ossos (n=5 para cada cadáver. Nas 200 amostras examinadas do homogeneizado restante (n=40 para cada cadáver, em apenas duas amostras de uma leira foram detectados esporos de C. botulinum tipo C, enquanto que todas foram negativas para a tentativa de detecção da toxina botulínica pelo bioensaio em camundongo. Da forma como foi avaliado o processo de pré-compostagem de bovinos mortos pela intoxicação botulínica não contribuiu para a proliferação de C. botulinum.Five carcasses of cattle that died from botulism were composted in individual isolated beds with the purpose of monitoring the effect on spores of Clostridium botulinum and

  7. [Botulinum toxin therapy for spasticity].

    Science.gov (United States)

    Masakado, Yoshihisa

    2014-09-01

    Botulinum toxin (BTX) administered as an adjunct to other interventions for spasticity can act as a useful and effective therapeutic tool for treating patients disabled by spasticity. Presence of other non-reflex motor disorders (muscle stiffness, shortness, and contracture) can complicate the clinical course and disturb rehabilitative process of patients with spasticity. Treatment of spasticity using BTX can improve paralysis by correcting muscular imbalance that follows these diseases. In patients with chronic severe spasticity, we also have to address unique and difficult-to-treat clinical conditions such as abnormal posture and movement disorders. The effectiveness of BTX in treating some of these conditions is discussed. Because patients with neurological disabilities can show complex dysfunctions, specific functional limitations, goals, and expected outcomes of treatment should be evaluated and discussed with the patient, family members, and caregivers, prior to initiating BTX therapy. BTX therapy might improve not only care, passive function, but also motor functions in these patients by supplementing intensive rehabilitation with repetitive transcranial magnetic stimulation, transcranial direct-current stimulation, peripheral electrical stimulation, muscle stretching, and other rehabilitation strategies.

  8. Zebrafish Sensitivity to Botulinum Neurotoxins

    Science.gov (United States)

    Chatla, Kamalakar; Gaunt, Patricia S.; Petrie-Hanson, Lora; Ford, Lorelei; Hanson, Larry A.

    2016-01-01

    Botulinum neurotoxins (BoNT) are the most potent known toxins. The mouse LD50 assay is the gold standard for testing BoNT potency, but is not sensitive enough to detect the extremely low levels of neurotoxin that may be present in the serum of sensitive animal species that are showing the effects of BoNT toxicity, such as channel catfish affected by visceral toxicosis of catfish. Since zebrafish are an important animal model for diverse biomedical and basic research, they are readily available and have defined genetic lines that facilitate reproducibility. This makes them attractive for use as an alternative bioassay organism. The utility of zebrafish as a bioassay model organism for BoNT was investigated. The 96 h median immobilizing doses of BoNT/A, BoNT/C, BoNT/E, and BoNT/F for adult male Tübingen strain zebrafish (0.32 g mean weight) at 25 °C were 16.31, 124.6, 4.7, and 0.61 picograms (pg)/fish, respectively. These findings support the use of the zebrafish-based bioassays for evaluating the presence of BoNT/A, BoNT/E, and BoNT/F. Evaluating the basis of the relatively high resistance of zebrafish to BoNT/C and the extreme sensitivity to BoNT/F may reveal unique functional patterns to the action of these neurotoxins. PMID:27153088

  9. Zebrafish Sensitivity to Botulinum Neurotoxins

    Directory of Open Access Journals (Sweden)

    Kamalakar Chatla

    2016-05-01

    Full Text Available Botulinum neurotoxins (BoNT are the most potent known toxins. The mouse LD50 assay is the gold standard for testing BoNT potency, but is not sensitive enough to detect the extremely low levels of neurotoxin that may be present in the serum of sensitive animal species that are showing the effects of BoNT toxicity, such as channel catfish affected by visceral toxicosis of catfish. Since zebrafish are an important animal model for diverse biomedical and basic research, they are readily available and have defined genetic lines that facilitate reproducibility. This makes them attractive for use as an alternative bioassay organism. The utility of zebrafish as a bioassay model organism for BoNT was investigated. The 96 h median immobilizing doses of BoNT/A, BoNT/C, BoNT/E, and BoNT/F for adult male Tübingen strain zebrafish (0.32 g mean weight at 25 °C were 16.31, 124.6, 4.7, and 0.61 picograms (pg/fish, respectively. These findings support the use of the zebrafish-based bioassays for evaluating the presence of BoNT/A, BoNT/E, and BoNT/F. Evaluating the basis of the relatively high resistance of zebrafish to BoNT/C and the extreme sensitivity to BoNT/F may reveal unique functional patterns to the action of these neurotoxins.

  10. Atypical Glycolysis in Clostridium thermocellum

    NARCIS (Netherlands)

    Zhou, J.; Olson, D.G.; Argyros, D.A.; Deng, Y.; Van Gulik, W.M.; Van Dijken, J.P.; Lynd, L.R.

    2013-01-01

    Cofactor specificities of glycolytic enzymes in Clostridium thermocellum were studied with cellobiose-grown cells from batch cultures. Intracellular glucose was phosphorylated by glucokinase using GTP rather than ATP. Although phosphofructokinase typically uses ATP as a phosphoryl donor, we found

  11. Regulation of toxin and bacteriocin gene expression in Clostridium by interchangeable RNA polymerase sigma factors.

    Science.gov (United States)

    Dupuy, Bruno; Raffestin, Stéphanie; Matamouros, Susana; Mani, Nagraj; Popoff, Michel R; Sonenshein, Abraham L

    2006-05-01

    The production of major extracellular toxins by pathogenic strains of Clostridium botulinum, Clostridium tetani and Clostridium difficile, and a bacteriocin by Clostridium perfringens is dependent on a related group of RNA polymerase sigma-factors. These sigma-factors (BotR, TetR, TcdR and UviA) were shown to be sufficiently similar that they could substitute for one another in in vitro DNA binding and run-off transcription experiments. In cells, however, the sigma-factors fell into two subclasses. BotR and TetR were able to direct transcription of their target genes in a fully reciprocal manner. Similarly, UviA and TcdR were fully interchangeable. Neither BotR nor TetR could substitute for UviA or TcdR, however, and neither UviA nor TcdR could direct transcription of the natural targets of BotR or TetR. The extent of functional interchangeability of the sigma-factors was attributed to the strong conservation of their subregion 4.2 sequences and the conserved -35 sequences of their target promoters, while restrictions on interchangeability were attributed to variations in their subregion 2.4 sequences and the target site -10 sequences. The four sigma-factors have been assigned to group 5 of the sigma(70) family and seem to have arisen from a common ancestral protein that may have co-evolved with the genes whose transcription they direct. A fifth Clostridiumsigma-factor, sigma(Y) of Clostridium acetobutylicum, resembles the TcdR family, but was not functionally interchangeable with members of this family.

  12. Diagnosis of Clostridium difficile

    DEFF Research Database (Denmark)

    Jensen, M B F; Olsen, K E P; Nielsen, X C

    2015-01-01

    The diagnosis of Clostridium difficile infection (CDI) requires the detection of toxigenic C. difficile or its toxins and a clinical assessment. We evaluated the performance of four nucleic acid amplification tests (NAATs) detecting toxigenic C. difficile directly from faeces compared to routine...... ribotyping and toxinotyping (TT) were performed on culture-positive samples. In parallel, the samples were analysed by four NAATs; two targeting tcdA or tcdB (illumigene® C. difficile and PCRFast® C. difficile A/B) and two multi-target real-time (RT) PCR assays also targeting cdt and tcdC alleles...... characteristic of epidemic and potentially more virulent PCR ribotypes 027, 066 and 078 (GeneXpert® C. difficile/Epi and an 'in-house RT PCR' two-step algorithm). The multi-target assays were significantly more sensitive compared to routine toxigenic culture (p 

  13. ADP-ribosylation of actins in fibroblasts and myofibroblasts by botulinum C2 toxin: Influence on microfilament morphology and migratory behavior

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Petersen, Ole William

    1996-01-01

    ., Petersen, O. W. J. Cell Biol. 1996, 134, 67-80). In the present study we have expanded on the functional significance of actin isotypes in fibroblasts from the opposite point of view, namely filamentous nonmuscle actin. Nonmuscle actins in fibroblasts and myofibroblasts were ADP-ribosylated by Clostridium...... botulinum C2 toxin. The substrate for C2 toxin is globular actin, which upon ribosylation cannot incorporate into microfilaments. The pattern of actin ADP-ribosylation in (myo)fibroblasts in the presence of [32P]NAD was analyzed by isoelectric focusing, fluorography and immunoblotting. The influence of C2...

  14. Botulinum Toxin in Neurogenic Detrusor Overactivity

    Directory of Open Access Journals (Sweden)

    Carlos Arturo Levi D'Ancona

    2012-09-01

    Full Text Available Purpose To evaluate the effects of botulinum toxin on urodynamic parameters and quality of life in patients with neurogenic detrusor overactivity. Methods Thirty four adult patients with spinal cord injury and detrusor overactivity were selected. The patients received 300 units of botulinum toxin type A. The endpoints evaluated with the episodes of urinary incontinence and measured the maximum cystometric capacity, maximum amplitude of detrusor pressure and bladder compliance at the beginning and end of the study (24 weeks and evaluated the quality of life by applying the Qualiveen questionnaire. Results A significant decrease in the episodes of urinary incontinence was observed. All urodynamic parameters presented a significant improvement. The same was observed in the quality of life index and the specific impact of urinary problems scores from the Qualiveen questionnaire. Six patients did not complete the study, two due to incomplete follow-up, and four violated protocol and were excluded from the analyses. No systemic adverse events of botulinum toxin type A were reported. Conclusions A botulinum toxin type A showed a significantly improved response in urodynamics parameters and specific and general quality of life.

  15. Surgery and botulinum toxin in congenital esotropia.

    Science.gov (United States)

    Ruiz, Miguel F; Alvarez, María T; Sánchez-Garrido, Carmen M; Hernáez, José M; Rodríguez, José M

    2004-10-01

    In a previous study we investigated the advantages and drawbacks of early and delayed injection of botulinum toxin as primary treatment of infantile esotropia with nystagmus in abduction (IENA). We carried out a further study to investigate the role and efficacy of surgery in this condition and to determine the possible effect of previous injection of both medial recti with botulinum toxin in patients requiring a final horizontal surgical correction. Review of the records of 44 patients (24 girls and 20 boys) with IENA seen between 1979 and 1998 who had undergone at least one horizontal surgical procedure. The outcomes in the 16 patients who had previously received botulinum toxin were compared with those in the 28 patients for whom surgery was the primary treatment. There was a negative correlation between the pretreatment esotropic angle and age (Pearson's r = -0.45, p IENA with delayed diagnosis and in cases associated with unsteadiness of binocular vision or with nonhorizontal deviations. Initial treatment with botulinum toxin, injected into both medial recti, is effective, reducing the amount of further horizontal surgery and favouring postoperative stability, except in children under 18 months, in whom injection of 5 units induces unbalanced dissociated vertical deviation.

  16. Outbreak of necrotizing fasciitis due to Clostridium sordellii among black-tar heroin users.

    Science.gov (United States)

    Kimura, Akiko C; Higa, Jeffrey I; Levin, Robert M; Simpson, Gail; Vargas, Yolanda; Vugia, Duc J

    2004-05-01

    In California, black tar heroin (BTH) use among injection drug users (IDUs) has resulted in an increased number of cases of wound botulism due to Clostridium botulinum, tetanus due to Clostridium tetani, and necrotizing soft-tissue infections due to a variety of clostridia. From December 1999 to April 2000, nine IDUs in Ventura County, California, developed necrotizing fasciitis; 4 died. Cultures of wound specimens from 6 case patients yielded Clostridium sordellii. Some of the patients appeared to have the toxic shock syndrome previously reported to be characteristic of toxin-mediated C. sordellii infection, which is characterized by hypotension, marked leukocytosis, and hemoconcentration. The suspected source of this outbreak was contaminated BTH that was injected subcutaneously or intramuscularly ("skin popped"). This outbreak of C. sordellii infection serves as another example of how BTH can potentially serve as a vehicle for transmitting severe and often deadly clostridial infections, and reinforces the need to educate IDUs and clinicians about the risks associated with skin popping of BTH.

  17. EtfA catalyses the formation of dipicolinic acid in Clostridium perfringens.

    Science.gov (United States)

    Orsburn, Benjamin C; Melville, Stephen B; Popham, David L

    2010-01-01

    Dipicolinic acid (DPA) is a major component of bacterial endospores, comprising 5-15% of the spore dry weight, and is important for spore stability and resistance properties. The biosynthetic precursor to DPA, dihydro-dipicolinic acid (DHDPA), is produced by DHDPA synthase within the lysine biosynthesis pathway. In Bacillus subtilis, and most other bacilli and clostridia, DHDPA is oxidized to DPA by the products of the spoVF operon. Analysis of the genomes of the clostridia in Cluster I, including the pathogens Clostridium perfringens, Clostridium botulinum and Clostridium tetani, has shown that no spoVF orthologues exist in these organisms. DPA synthase was purified from extracts of sporulating C. perfringens cells. Peptide sequencing identified an electron transfer flavoprotein, EtfA, in this purified protein fraction. A C. perfringens strain with etfA inactivated is blocked in late stage sporulation and produces < or = 11% of wild-type DPA levels. C. perfringens EtfA was expressed in and purified from Escherichia coli, and this protein catalysed DPA formation in vitro. The sequential production of DHDPA and DPA in C. perfringens appears to be catalysed by DHDPA synthase followed by EtfA. Genome sequence data and the taxonomy of spore-forming species suggest that this may be the ancestral mechanism for DPA synthesis.

  18. Proposal to restrict the genus Clostridium Prazmowski to Clostridium butyricum and related species.

    Science.gov (United States)

    Lawson, Paul A; Rainey, Fred A

    2016-02-01

    The genus Clostridium as presently constituted is phylogenetically and phenotypically incoherent. Data from polyphasic taxonomic studies indicate that the genus comprises a collection of very heterogeneous species. Numerous phylogenetic studies, principally based on sequencing of the 16S rRNA gene, indicate that the genus Clostridium should be restricted to Clostridium cluster I as Clostridium sensu stricto . Despite these findings, authors continue to add novel species to the genus Clostridium that do not fall within the radiation of cluster I and the type species Clostridium butyricum , thus perpetuating the confusion associated with the taxonomy of this group. Here, we formally propose that members of the genus Clostridium Prazmowski be restricted to the type species C. butyricum and cluster I species. Eubacterium moniliforme , Eubacterium tarantellae , Sarcina maxima and Sarcina ventriculi should be transferred to the genus Clostridium as Clostridium moniliforme comb. nov., Clostridium tarantellae comb. nov., Clostridium maximum comb. nov. and Clostridium ventriculi comb. nov. A novel genus, Hathewaya gen. nov., is proposed for the species Clostridium histolyticum , Clostridium limosum and Clostridium proteolyticum as Hathewaya histolytica gen. nov. comb. nov., Hathewaya limosa comb. nov. and Hathewaya proteolytica comb. nov. The type species of the genus Hathewaya is Hathewaya histolytica.

  19. Beneficial effects of botulinum toxin type A in trigeminal neuralgia

    OpenAIRE

    Zúñiga,Carlos; Díaz,Sergio; Piedimonte,Fabián; Micheli,Federico

    2008-01-01

    Botulinum toxin has been thoroughly studied as a potential tool in the treatment of several pain syndromes. Therefore, we assessed the clinical effects of botulinum toxin type A injections in 12 patients with otherwise unresponsive idiopathic trigeminal neuralgia. Patients were infiltrated with 20-50 units of botulinum toxin in trigger zones. Those who presented with mandibular involvement were also infiltrated in the masseter muscle. The patients were assessed on a weekly basis using the Vis...

  20. Multiplex PCR for identification of Clostridium chauvoei and Clostridium septicum

    OpenAIRE

    Assis, R.A.; Lobato, F.C.F.; Lobato, Z.I.P.; Camargos, M.F.; Nascimento, R.A.P.; Vargas, A.P.C.; Salvarani, F.M.; Uzal, F.A.

    2008-01-01

    Padronizou-se uma técnica de reação em cadeia da polimerase múltipla (PCR multiplex) para detecção de Clostridium chauvoei e Clostridium septicum em culturas puras. Foram utilizados pares de iniciadores para segmentos específicos dos genes que codificam a flagelina de C. chauvoei e a toxina alfa de C. septicum. Para avaliaçã o da PCR multiplex, foram testados 16 isolados clínicos de C. chauvoei e 15 isolados de C. septicum provenientes de ruminantes, quatro sementes vacinais de cada um desses...

  1. Genomics of Clostridium tetani.

    Science.gov (United States)

    Brüggemann, Holger; Brzuszkiewicz, Elzbieta; Chapeton-Montes, Diana; Plourde, Lucile; Speck, Denis; Popoff, Michel R

    2015-05-01

    Genomic information about Clostridium tetani, the causative agent of the tetanus disease, is scarce. The genome of strain E88, a strain used in vaccine production, was sequenced about 10 years ago. One additional genome (strain 12124569) has recently been released. Here we report three new genomes of C. tetani and describe major differences among all five C. tetani genomes. They all harbor tetanus-toxin-encoding plasmids that contain highly conserved genes for TeNT (tetanus toxin), TetR (transcriptional regulator of TeNT) and ColT (collagenase), but substantially differ in other plasmid regions. The chromosomes share a large core genome that contains about 85% of all genes of a given chromosome. The non-core chromosome comprises mainly prophage-like genomic regions and genes encoding environmental interaction and defense functions (e.g. surface proteins, restriction-modification systems, toxin-antitoxin systems, CRISPR/Cas systems) and other fitness functions (e.g. transport systems, metabolic activities). This new genome information will help to assess the level of genome plasticity of the species C. tetani and provide the basis for detailed comparative studies. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  2. Emerging opportunities for serotypes of botulinum neurotoxins.

    Science.gov (United States)

    Peng Chen, Zhongxing; Morris, J Glenn; Rodriguez, Ramon L; Shukla, Aparna Wagle; Tapia-Núñez, John; Okun, Michael S

    2012-11-07

    Two decades ago, botulinum neurotoxin (BoNT) type A was introduced to the commercial market. Subsequently, the toxin was approved by the FDA to address several neurological syndromes, involving muscle, nerve, and gland hyperactivity. These syndromes have typically been associated with abnormalities in cholinergic transmission. Despite the multiplicity of botulinal serotypes (designated as types A through G), therapeutic preparations are currently only available for BoNT types A and B. However, other BoNT serotypes are under study for possible clinical use and new clinical indications; To review the current research on botulinum neurotoxin serotypes A-G, and to analyze potential applications within basic science and clinical settings; The increasing understanding of botulinal neurotoxin pathophysiology, including the neurotoxin's effects on specific neuronal populations, will help us in tailoring treatments for specific diagnoses, symptoms and patients. Scientists and clinicians should be aware of the full range of available data involving neurotoxin subtypes A-G.

  3. Autism and Clostridium tetani.

    Science.gov (United States)

    Bolte, E R

    1998-08-01

    Autism is a severe developmental disability believed to have multiple etiologies. This paper outlines the possibility of a subacute, chronic tetanus infection of the intestinal tract as the underlying cause for symptoms of autism observed in some individuals. A significant percentage of individuals with autism have a history of extensive antibiotic use. Oral antibiotics significantly disrupt protective intestinal microbiota, creating a favorable environment for colonization by opportunistic pathogens. Clostridium tetani is an ubiquitous anaerobic bacillus that produces a potent neurotoxin. Intestinal colonization by C. tetani, and subsequent neurotoxin release, have been demonstrated in laboratory animals which were fed vegetative cells. The vagus nerve is capable of transporting tetanus neurotoxin (TeNT) and provides a route of ascent from the intestinal tract to the CNS. This route bypasses TeNT's normal preferential binding sites in the spinal cord, and therefore the symptoms of a typical tetanus infection are not evident. Once in the brain, TeNT disrupts the release of neurotransmitters by the proteolytic cleavage of synaptobrevin, a synaptic vesicle membrane protein. This inhibition of neurotransmitter release would explain a wide variety of behavioral deficits apparent in autism. Lab animals injected in the brain with TeNT have exhibited many of these behaviors. Some children with autism have also shown a significant reduction in stereotyped behaviors when treated with antimicrobials effective against intestinal clostridia. When viewed as sequelae to a subacute, chronic tetanus infection, many of the puzzling abnormalities of autism have a logical basis. A review of atypical tetanus cases, and strategies to test the validity of this paper's hypothesis, are included.

  4. Protein Receptor(s) of Botulinum Neurotoxin

    Science.gov (United States)

    2005-01-01

    B cells (strain Okra) in a 1.5 ml microcentrifuge tube kept in -80’C was added to a 10 ml cooked meat medium (Difco Laboratories, Becton Dickinson, MD...and cultured at 30 TC for 18 to 20 hours. The C. botulinum type B cells in the cooked meat medium was inoculated in 500 ml of toxin production...drugs, carcinogens and decreasing stress of xenobiotic compounds, intracellular carrier proteins and reduction of hydroxyperoxides and nitrates. Because

  5. Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathryn L.; Marks, James D.; Varnum, Susan M.

    2012-11-15

    Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the present study, we have developed an ELISA-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotype A, B, C, D, E and F. With engineered high-affinity antibodies, the assays have sensitivities in buffer of 8 fM (1.2 pg/mL) for serotypes A and B, and 32 fM (4.9 pg/mL) for serotypes C, D, E, and F. Using clinical and environmental samples (serum and milk), the microarray is capable of detecting BoNT/A-F to the same levels as in standard buffer. Cross reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical or environmental samples.

  6. Clostridium difficile Infection in Outpatients

    Centers for Disease Control (CDC) Podcasts

    2011-11-07

    Dr. Jon Mark Hirshon, Associate Professor of Emergency Medicine at the University of Maryland School of Medicine, discusses Clostridium difficile infection in outpatients.  Created: 11/7/2011 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 11/21/2011.

  7. The potential role of Clostridium botulinum toxin in the treatment of equine laminitis

    NARCIS (Netherlands)

    Hardeman, L C

    2016-01-01

    Laminitis is a severe and painful disease in the horse that may lead to loosening of the attachment of the distal phalanx to the horny hoof capsule. Although a large variety of treatment options has come and gone since the first descriptions of laminitis, there is certainly a thus far unmet need for

  8. SELECTIVE ISOLATION OF CLOSTRIDIUM BOTULINUM DNA FROM VEGETATIVE CELLS IN SEDIMENTS. (R826552)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  9. Botulinum Toxin Type A as a Therapeutic Agent against Headache and Related Disorders

    Directory of Open Access Journals (Sweden)

    Siro Luvisetto

    2015-09-01

    Full Text Available Botulinum neurotoxin A (BoNT/A is a toxin produced by the naturally-occurring Clostridium botulinum that causes botulism. The potential of BoNT/A as a useful medical intervention was discovered by scientists developing a vaccine to protect against botulism. They found that, when injected into a muscle, BoNT/A causes a flaccid paralysis. Following this discovery, BoNT/A has been used for many years in the treatment of conditions of pathological muscle hyperactivity, like dystonias and spasticities. In parallel, the toxin has become a “glamour” drug due to its power to ward off facial wrinkles, particularly frontal, due to the activity of the mimic muscles. After the discovery that the drug also appeared to have a preventive effect on headache, scientists spent many efforts to study the potentially-therapeutic action of BoNT/A against pain. BoNT/A is effective at reducing pain in a number of disease states, including cervical dystonia, neuropathic pain, lower back pain, spasticity, myofascial pain and bladder pain. In 2010, regulatory approval for the treatment of chronic migraine with BoNT/A was given, notwithstanding the fact that the mechanism of action is still not completely elucidated. In the present review, we summarize experimental evidence that may help to clarify the mechanisms of action of BoNT/A in relation to the alleviation of headache pain, with particular emphasis on preclinical studies, both in animals and humans. Moreover, we summarize the latest clinical trials that show evidence on headache conditions that may obtain benefits from therapy with BoNT/A.

  10. Botulinum toxin type A products are not interchangeable: a review of the evidence

    Directory of Open Access Journals (Sweden)

    Brin MF

    2014-10-01

    Full Text Available Mitchell F Brin,1,2 Charmaine James,3 John Maltman1 1Allergan, Inc., Irvine, CA, USA; 2Department of Neurology, University of California, Irvine, CA, USA; 3Allergan, Marlow, UKAbstract: Botulinum toxin type A (BoNTA products are injectable biologic medications derived from Clostridium botulinum bacteria. Several different BoNTA products are marketed in various countries, and they are not interchangeable. Differences between products include manufacturing processes, formulations, and the assay methods used to determine units of biological activity. These differences result in a specific set of interactions between each BoNTA product and the tissue injected. Consequently, the products show differences in their in vivo profiles, including preclinical dose response curves and clinical dosing, efficacy, duration, and safety/adverse events. Most, but not all, published studies document these differences, suggesting that individual BoNTA products act differently depending on experimental and clinical conditions, and these differences may not always be predictable. Differentiation through regulatory approvals provides a measure of confidence in safety and efficacy at the specified doses for each approved indication. Moreover, the products differ in the amount of study to which they have been subjected, as evidenced by the number of publications in the peer-reviewed literature and the quantity and quality of clinical studies. Given that BoNTAs are potent biological products that meet important clinical needs, it is critical to recognize that their dosing and product performance are not interchangeable and each product should be used according to manufacturer guidelines.Keywords: onabotulinumtoxinA, abobotulinumtoxinA, incobotulinumtoxinA, glabellar lines, non-interchangeability

  11. An evidence-based review of botulinum toxin (Botox) applications in non-cosmetic head and neck conditions

    Science.gov (United States)

    Persaud, Ricardo; Garas, George; Silva, Sanjeev; Stamatoglou, Constantine; Chatrath, Paul; Patel, Kalpesh

    2013-01-01

    Botulinum toxin (Botox) is an exotoxin produced from Clostridium botulinum. It works by blocking the release of acetylcholine from the cholinergic nerve end plates leading to inactivity of the muscles or glands innervated. Botox is best known for its beneficial role in facial aesthetics but recent literature has highlighted its usage in multiple non-cosmetic medical and surgical conditions. This article reviews the current evidence pertaining to Botox use in the head and neck. A literature review was conducted using The Cochrane Controlled Trials Register, Medline and EMBASE databases limited to English Language articles published from 1980 to 2012. The findings suggest that there is level 1 evidence supporting the efficacy of Botox in the treatment of spasmodic dysphonia, essential voice tremor, headache, cervical dystonia, masticatory myalgia, sialorrhoea, temporomandibular joint disorders, bruxism, blepharospasm, hemifacial spasm and rhinitis. For chronic neck pain there is level 1 evidence to show that Botox is ineffective. Level 2 evidence exists for vocal tics, trigeminal neuralgia, dysphagia and post-laryngectomy oesophageal speech. For stuttering, ‘first bite syndrome’, facial nerve paresis, Frey's syndrome, oromandibular dystonia and palatal/stapedial myoclonus the evidence is level 4. Thus, the literature highlights a therapeutic role for Botox in a wide range of non-cosmetic conditions pertaining to the head and neck (mainly level 1 evidence). With ongoing research, the spectrum of clinical applications and number of people receiving Botox will no doubt increase. Botox appears to justify its title as ‘the poison that heals’. PMID:23476731

  12. PEG precipitation coupled with chromatography is a new and sufficient method for the purification of botulinum neurotoxin type B [corrected].

    Directory of Open Access Journals (Sweden)

    Yao Zhao

    Full Text Available Clostridium botulinum neurotoxins are used to treat a variety of neuro-muscular disorders, as well as in cosmetology. The increased demand requires efficient methods for the production and purification of these toxins. In this study, a new purification process was developed for purifying type B neurotoxin. The kinetics of C.botulinum strain growth and neurotoxin production were determined for maximum yield of toxin. The neurotoxin was purified by polyethylene glycol (PEG precipitation and chromatography. Based on design of full factorial experiment, 20% (w/v PEG-6000, 4 °C, pH 5.0 and 0.3 M NaCl were optimal conditions to obtain a high recovery rate of 87% for the type B neurotoxin complex, as indicated by a purification factor of 61.5 fold. Furthermore, residual bacterial cells, impurity proteins and some nucleic acids were removed by PEG precipitation. The following purification of neurotoxin was accomplished by two chromatography techniques using Sephacryl™ S-100 and phenyl HP columns. The neurotoxin was recovered with an overall yield of 21.5% and the purification factor increased to 216.7 fold. In addition, a mouse bioassay determined the purified neurotoxin complex possessed a specific toxicity (LD(50 of 4.095 ng/kg.

  13. The therapeutic usage of botulinum toxin (Botox in non-cosmetic head and neck conditions – An evidence based review

    Directory of Open Access Journals (Sweden)

    Kamran Habib Awan

    2017-01-01

    Full Text Available Botulinum toxin (Botox is an exotoxin produced from Clostridium botulinum. It blocks the release of acetylcholine from the cholinergic nerve end plates resulting in inactivity of the muscles or glands innervated. The efficacy of Botox in facial aesthetics is well established; however, recent literature has highlighted its utilization in multiple non-cosmetic medical and surgical conditions. The present article reviews the current evidence pertaining to Botox use in the non-cosmetic head and neck conditions. A literature search was conducted using MEDLINE, EMBASE, ISI Web of Science and the Cochrane databases limited to English Language articles published from January 1980 to December 2014. The findings showed that there is level 1 evidence supporting the efficacy of Botox in the treatment of laryngeal dystonia, headache, cervical dystonia, masticatory myalgia, sialorrhoea, temporomandibular joint disorders, bruxism, blepharospasm, hemifacial spasm and rhinitis. For chronic neck pain there is level 1 evidence to show that Botox is ineffective. Level 2 evidence exists for vocal tics and trigeminal. For stuttering, facial nerve paresis, Frey’s syndrome and oromandibular dystonia the evidence is level 4. Thus, there is compelling evidence in the published literature to demonstrate the beneficial role of Botox in a wide range of non-cosmetic conditions pertaining to the head and neck (mainly level 1 evidence. With more and more research, the range of clinical applications and number of individuals getting Botox will doubtlessly increase. Botox appears to justify its title as ‘the poison that heals’.

  14. Long lasting dysautonomia due to botulinum toxin B poisoning: clinical-laboratory follow up and difficulties in initial diagnosis.

    Science.gov (United States)

    Potulska-Chromik, Anna; Zakrzewska-Pniewska, Beata; Szmidt-Sałkowska, Elżbieta; Lewandowski, Jacek; Siński, Maciej; Przyjałkowski, Witold; Kostera-Pruszczyk, Anna

    2013-10-30

    Botulism is an acute form of poisoning caused by one of four types (A, B, E, F) toxins produced by Clostridium botulinum, ananaerobic, spore forming bacillus. Usually diagnosis of botulism is considered in patients with predominant motor symptoms: muscle weakness with intact sensation and preserved mental function. We report a case of 56-year-old Caucasian female with a history of arterial hypertension, who presented with acute respiratory failure and bilateral ptosis misdiagnosed as brainstem ischemia. She had severe external and internal ophtalmoplegia, and autonomic dysfunction with neither motor nor sensory symptoms from upper and lower limbs. Diagnosis of botulinum toxin poisoning was made and confirmed by serum antibody testing in the mouse inoculation test. Ophtalmoplegia, autonomic dysfunction and respiratory failure can be caused by botulism. Early treatment and intensive care is essential for survival and recovery. The electrophysiological tests are crucial to correct and rapid diagnosis. Botulism (especially type B) should be considered in any case of acute or predominant isolated autonomic dysfunction.

  15. Sequencing and phylogenetic analysis of neurotoxin gene from an environmental isolate of Clostridium sp.: comparison with other clostridial neurotoxins.

    Science.gov (United States)

    Dixit, Aparna; Alam, Syed Imteyaz; Singh, Lokendra

    2006-07-01

    A Clostridium sp. isolated from intestine of decaying fish exhibited 99% sequence identity with C. tetani at 16S rRNA level. It produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) as well as tetanus antitoxin. The gene fragments for light chain, C-terminal and N-terminal regions of the heavy chain of the toxin were amplified using three reported primer sets for tetanus neurotoxin (TeNT). The neurotoxin gene fragments were cloned in Escherichia coli and sequenced. The sequences obtained exhibited approximately 98, 99 and 98% sequence identity with reported gene sequences of TeNT/LC, TeNT/HC and TeNT/HN, respectively. The phylogenetic interrelationship between the neurotoxin gene of Clostridium sp. with previously reported gene sequences of Clostridium botulinum A to G and C. tetani was examined by analysis of differences in the nucleotide sequences. Six amino acids were substituted at four different positions in the light chain of neurotoxin from the isolate when compared with the reported closest sequence of TeNT. Of these, four were located in the beta15 motif at a solvent inaccessible, buried region of the protein molecule. One of these substitutions were on the solvent accessible surface residue of alpha1 motif, previously shown to have strong sequence conservation. A substitution of two amino acids observed in N-terminal region of heavy chain were buried residues, located in the beta21 and beta37 motifs showing variability in other related sequences. The C-terminal region responsible for binding to receptor was conserved, showing no changes in the amino acid sequence.

  16. A historical and proteomic analysis of botulinum neurotoxin type/G

    Directory of Open Access Journals (Sweden)

    Rees Jon

    2011-10-01

    Full Text Available Abstract Background Clostridium botulinum is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs. There are seven known serotypes of BoNTs (/A through/G, all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs, we conducted an in silico and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity in vitro. In addition, we review previous literature to provide a complete description of the BoNT/G complex. Results An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT and three NAPs--nontoxic-nonhemagglutinin (NTNH and two hemagglutinins (HA70 and HA17. Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison. Conclusions The in silico protein sequence comparisons established that the/G complex is phenetically related to the other six serotypes of C. botulinum. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and NAPs, along with the activity of the commercial/G complex. The use of data

  17. Ultrasound-guided botulinum toxin injections

    Directory of Open Access Journals (Sweden)

    S. E. Khatkova

    2016-01-01

    Full Text Available One of the key conditions for achieving the desirable result during botulinum toxin therapy for muscular dystonia, spasticity, and other diseases accompanied by spasm, pain, and autonomic dysfunction (dystonias, spasticity, etc. is the proper administration of the agent into the muscles directly involved in the pathological process. The exact entry of botulinum toxin into the target muscles is essential for successful and safe treatment because its injection into a normal muscle may cause side effects. The most common errors are the incorrect depth and incorrect direction of a needle on insertion. Therefore, the exact injection of the agent particularly into the shallow and deep muscles is a difficult task even for an experienced specialist and requires the use of controlling methods.The European Consensus on Botulinum Toxin Therapy points out that various injection techniques are needed for the better identification of necessary muscles. However, there are currently no reports on the clear advantage of any technique. In our country, injections using palpation and anatomical landmarks have been widely used in routine practice so far; electromyographic monitoring and electrostimulation have been less frequently applied. In recent years, the new method ultrasound-guided injection has continued to grow more popular. This effective, accessible, and easy-to-use method makes it possible to manage a real-time injection process and to ensure the exact entry of the agent into the muscle. This paper is dedicated to a comparative analysis of different injection methods and to a description of the ultrasound-guided technique and its advantages over others. 

  18. Botulinum toxin for treatment of glandular hypersecretory disorders.

    LENUS (Irish Health Repository)

    Laing, T A

    2012-02-03

    SUMMARY: The use of botulinum toxin to treat disorders of the salivary glands is increasing in popularity in recent years. Recent reports of the use of botulinum toxin in glandular hypersecretion suggest overall favourable results with minimal side-effects. However, few randomised clinical trials means that data are limited with respect to candidate suitability, treatment dosages, frequency and duration of treatment. We report a selection of such cases from our own department managed with botulinum toxin and review the current data on use of the toxin to treat salivary gland disorders such as Frey\\'s syndrome, excessive salivation (sialorrhoea), focal and general hyperhidrosis, excessive lacrimation and chronic rhinitis.

  19. Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene.

    Science.gov (United States)

    Sasaki, Yoshimasa; Kojima, Akemi; Aoki, Hiroshi; Ogikubo, Yasuaki; Takikawa, Noriyasu; Tamura, Yutaka

    2002-05-01

    The flagellin genes (fliC) of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum were analysed by PCR amplification and DNA sequencing. The five Clostridium species have at least two copies of the flagellin gene (fliC) arranged in tandem on the chromosome. The deduced N- and C-terminal aminoacid sequences of the flagellin proteins (FliCs) of these clostridia are well conserved but their central region aminoacid sequences are not. Phylogenic analysis based on the N-terminal aminoacid sequence of the FliC protein revealed that these clostridia, which belong to Clostridium 16S rDNA phylogenic cluster I (), are more closely related to Bacillus subtilis than to Clostridium difficile, which belongs to the cluster XI. Moreover, a multiplex polymerase reaction (PCR) system based on the fliC sequence was developed to rapidly identify C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. PCR of each Clostridium amplified a species-specific band. The multiplex PCR system may be useful for rapid identification of pathogenic clostridia.

  20. 9 CFR 113.107 - Clostridium Haemolyticum Bacterin.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Clostridium Haemolyticum Bacterin. 113... REQUIREMENTS Inactivated Bacterial Products § 113.107 Clostridium Haemolyticum Bacterin. Clostridium Haemolyticum Bacterin shall be produced from a culture of Clostridium haemolyticum which has been inactivated...

  1. Identification of a Botulinum Neurotoxin-like Toxin in a Commensal Strain of Enterococcus faecium.

    Science.gov (United States)

    Zhang, Sicai; Lebreton, Francois; Mansfield, Michael J; Miyashita, Shin-Ichiro; Zhang, Jie; Schwartzman, Julia A; Tao, Liang; Masuyer, Geoffrey; Martínez-Carranza, Markel; Stenmark, Pål; Gilmore, Michael S; Doxey, Andrew C; Dong, Min

    2018-02-14

    Botulinum neurotoxins (BoNTs), produced by various Clostridium strains, are a family of potent bacterial toxins and potential bioterrorism agents. Here we report that an Enterococcus faecium strain isolated from cow feces carries a BoNT-like toxin, designated BoNT/En. It cleaves both VAMP2 and SNAP-25, proteins that mediate synaptic vesicle exocytosis in neurons, at sites distinct from known BoNT cleavage sites on these two proteins. Comparative genomic analysis determines that the E. faecium strain carrying BoNT/En is a commensal type and that the BoNT/En gene is located within a typical BoNT gene cluster on a 206 kb putatively conjugative plasmid. Although the host species targeted by BoNT/En remains to be determined, these findings establish an extended member of BoNTs and demonstrate the capability of E. faecium, a commensal organism ubiquitous in humans and animals and a leading cause of hospital-acquired multi-drug-resistant (MDR) infections, to horizontally acquire, and possibly disseminate, a unique BoNT gene cluster. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Viability of Clostridium sporogenes spores after CaO hygienization of meat waste

    Directory of Open Access Journals (Sweden)

    Justyna Bauza-Kaszewska

    2014-09-01

    Full Text Available The occurrence of the pathogenic species [i]C. perfringens[/i] and [i]C. botulinum spores[/i] in animal by-products poses a potential epidemiological hazard. Strong entero- and neurotoxins produced by these bacteria adversely affect human health. To inactivate pathogens present in animal by-products, waste must be subjected to various methods of sanitization. The aim of the presented study was to estimate the effect of different doses of CaO on the viability of spores [i] Clostridium sporogenes[/i] in meat wastes category 3. During the research, two doses of burnt lime were added to the poultry mince meat and meat mixed with swine blood contaminated with [i]Clostridium sporogenes[/i] spore suspension. Half of the samples collected for microbiological analyses were buffered to achieve the pH level ~7, the other were examined without pH neutralization. To estimate the spore number, 10-fold dilution series in peptone water was prepared and heat-treated at 80 °C for 10 min. After cooling-down, one milliliter of each dilution was pour-plated onto DRCM medium solidified with agar. Statistical analysis were performed using the Statistica software. Application of 70% CaO caused complete inactivation of [i]Clostridium spores[/i] in meat wastes after 48 hours. The highest temperature achieved during the experiment was 67 °C. Rapid alkalization of the biomass resulted in increasing pH to values exceeding 12. The effect of liming was not dependent on the meat wastes composition nor CaO dose. The experiment proved the efficiency of liming as a method of animal by-products sanitization. Application of the obtained results may help reduce the epidemiological risk and ensure safety to people handling meat wastes at each stage of their processing and utilization.

  3. Persistence of Upper Blepharoptosis After Cosmetic Botulinum Toxin Type A.

    Science.gov (United States)

    Steinsapir, Kenneth D; Groth, Michael J; Boxrud, Cynthia A

    2015-07-01

    Upper eyelid ptosis after cosmetic botulinum toxin is generally considered short-lived and responsive to apraclonidine ophthalmic drops. The authors present a series with persistent ptosis. To report a series of patients with persistent upper eyelid ptosis after cosmetic botulinum toxin. A retrospective case review series of 7 patients referred for management after developing visually significant upper eyelid ptosis after cosmetic botulinum toxin type A treatment. Patients in this series experienced persistent visually significant ptosis after cosmetic botulinum toxin lasting from 6 weeks to 13 months. Six of the 7 patients were treated with apraclonidine ophthalmic solution. Apraclonidine drops appeared to be clinically effective within 4 to 6 weeks of the resolution of ptosis. Upper eyelid ptosis after cosmetic botulinum toxin can persist for many months after treatment. Based on this series, the authors propose that apraclonidine drops can be used at the time of initial assessment to predict the relative longevity of ptosis after cosmetic botulinum toxin treatment (Level 4 evidence recommendation). After a 1-week trial, responders can be advised that ptosis is likely to resolve in 4 to 6 weeks. Nonresponders should be counseled that resolution may take longer than 6 weeks.

  4. Botulinum toxin A for the Treatment of Overactive Bladder

    Directory of Open Access Journals (Sweden)

    Po-Fan Hsieh

    2016-02-01

    Full Text Available The standard treatment for overactive bladder starts with patient education and behavior therapies, followed by antimuscarinic agents. For patients with urgency urinary incontinence refractory to antimuscarinic therapy, currently both American Urological Association (AUA and European Association of Urology (EAU guidelines suggested that intravesical injection of botulinum toxin A should be offered. The mechanism of botulinum toxin A includes inhibition of vesicular release of neurotransmitters and the axonal expression of capsaicin and purinergic receptors in the suburothelium, as well as attenuation of central sensitization. Multiple randomized, placebo-controlled trials demonstrated that botulinum toxin A to be an effective treatment for patients with refractory idiopathic or neurogenic detrusor overactivity. The urinary incontinence episodes, maximum cystometric capacity, and maximum detrusor pressure were improved greater by botulinum toxin A compared to placebo. The adverse effects of botulinum toxin A, such as urinary retention and urinary tract infection, were primarily localized to the lower urinary tract. Therefore, botulinum toxin A offers an effective treatment option for patients with refractory overactive bladder.

  5. Botulinum toxin injection in laryngeal dyspnea.

    Science.gov (United States)

    Woisard, Virginie; Liu, Xuelai; Bes, Marie Christine Arné; Simonetta-Moreau, Marion

    2017-02-01

    Data, regarding the use of botulinum toxin (BT-A) in laryngeal dyspnea, are scarce, coming from some cases reports in the literature, including Vocal fold paralysis, laryngeal dystonia, vocal cord dysfunction also called paradoxical motion of the vocal fold (PMVF), and post-neuroleptic laryngeal dyskinesia. There is no consensus regarding the muscles and the doses to inject. The aim of this study is to present a retrospective review of patients treated in our ENT Department by BT-A injection in this indication. This study is a retrospective study describing patients who underwent an injection of botulinum toxin for laryngeal dyspnea in the ENT Department from 2005 to 2015 years. The inclusion criteria were a dyspnea associated with a laryngeal dysfunction, confirmed by flexible fiberoptic nasopharyngolaryngoscopy. Information concerning the causes of the dyspnea, the botulinum toxin BT-A injections procedure, post-injection follow-up, and respiratory outcome were collected for all patients included. In the group of 13 patients included, the main cause identified as principal factor linked with the short breath was: a bilateral VF paralysis (Patel et al., Otolaryngol Head Neck Surg 130:686-689, 7), laryngeal dystonia (Balkissoon and Kenn, Semin Respir Crit Care Med 33:595-605, 2), Anxiety syndrome associated with unilateral vocal fold paralysis or asthma (Marcinow et al., Laryngoscope 124:1425-1430, 3), and an isolated asthma (Zwirner et al., Eur Arch Otorhinolaryngol 254:242-245, 1). Nine out of the thirteen patients were improved by the injections. A BT-A-induced stable benefit for four patients led them to stop the injections in the follow-up. Good outcome was observed in five other patients (main cause: bilateral VP paralysis), allowing a progressive lengthening of the delay between BT-A injections. Four patients did not report a positive risk/benefit ratio after BT-A injections; two of them (with bilateral VF paralysis), because of respiratory side effects and

  6. [Variable forms of Clostridium felsineum].

    Science.gov (United States)

    Avrova, N P

    1977-01-01

    Variants of five types, differing in morphology of their colonies, resulted from spontaneous variability of Clostridium felsineum, the main agent involved in flax retting, when it was grown in a liquid medium and then on a solid medium. Morphological, cultural, and biochemical properties of the variants are described. The variants of the second and third types were less stable than those of the first type in the course of passages on growth media. The rate of spore formation was higher in the variants of the first type whereas the activity of pectolytic enzymes was greater in the variants of the second type.

  7. Clostridium difficile in Retail Meats

    Centers for Disease Control (CDC) Podcasts

    2009-04-16

    Clostridium difficile is a common cause of diarrhea in healthcare settings but little is known about what causes cases in the community. In this podcast, CDC's Dr. L. Clifford McDonald discusses two papers in the May 2009 edition of Emerging Infectious Diseases that explore whether the organism could be found in meat samples purchased in grocery stores in Arizona and Canada.  Created: 4/16/2009 by Emerging Infectious Diseases.   Date Released: 4/16/2009.

  8. TetR is a positive regulator of the tetanus toxin gene in Clostridium tetani and is homologous to botR.

    Science.gov (United States)

    Marvaud, J C; Eisel, U; Binz, T; Niemann, H; Popoff, M R

    1998-12-01

    The TetR gene immediately upstream from the tetanus toxin (TeTx) gene was characterized. It encodes a 21,562-Da protein which is related (50 to 65% identity) to the equivalent genes (botR) in Clostridium botulinum. TetR has the feature of a DNA binding protein with a basic pI (9.53). It contains a helix-turn-helix motif and shows 29% identity with other putative regulatory genes in Clostridium, i.e., uviA from C. perfringens and txeR from C. difficile. We report for the first time the transformation of C. tetani by electroporation, which permitted us to investigate the function of tetR. Overexpression of tetR in C. tetani induced an increase in TeTx production and in the level of the corresponding mRNA. This indicates that TetR is a transcriptional activator of the TeTx gene. Overexpression of botR/A (60% identity with TetR at the amino acid level) in C. tetani induced an increase in TeTx production comparable to that for overexpression of tetR. However, botR/C (50% identity with TetR at the amino acid level) was less efficient. This supports that TetR positively regulates the TeTx gene in C. tetani and that a conserved mechanism of regulation of the neurotoxin genes is involved in C. tetani and C. botulinum.

  9. Ultrasound Guidance for Botulinum Neurotoxin Chemodenervation Procedures.

    Science.gov (United States)

    Alter, Katharine E; Karp, Barbara I

    2017-12-28

    Injections of botulinum neurotoxins (BoNTs) are prescribed by clinicians for a variety of disorders that cause over-activity of muscles; glands; pain and other structures. Accurately targeting the structure for injection is one of the principle goals when performing BoNTs procedures. Traditionally; injections have been guided by anatomic landmarks; palpation; range of motion; electromyography or electrical stimulation. Ultrasound (US) based imaging based guidance overcomes some of the limitations of traditional techniques. US and/or US combined with traditional guidance techniques is utilized and or recommended by many expert clinicians; authors and in practice guidelines by professional academies. This article reviews the advantages and disadvantages of available guidance techniques including US as well as technical aspects of US guidance and a focused literature review related to US guidance for chemodenervation procedures including BoNTs injection.

  10. Treatment diary for botulinum toxin spasticity treatment

    DEFF Research Database (Denmark)

    Biering-Sørensen, Bo; Iversen, Helle K; Frederiksen, Inge M S

    2017-01-01

    The aim of this study is to develop a treatment diary for patients receiving spasticity treatment including botulinum toxin injection and physiotherapy and/or occupational therapy. The diary focuses on problems triggered by skeletal muscle overactivity; agreed goals for treatment and the patient......'s self-evaluation of achievement on the Goal Attainment Scale; which skeletal muscles were injected; physiotherapists' and occupational therapists' evaluation of the patients' achievement of objectives on the Goal Attainment Scale; and proposals for optimization of treatment and changing goals....... The evaluation included a satisfaction questionnaire and the WHO-QoL BREF and WHO-5 well-being score. Overall, 10 patients were enrolled in the pilot study. The patients were generally satisfied with the diary, found that it involved them more in their treatment and made it easier to set personal goals...

  11. Treatment diary for botulinum toxin spasticity treatment

    DEFF Research Database (Denmark)

    Biering-Sørensen, Bo; Iversen, Helle K; Frederiksen, Inge M S

    2017-01-01

    's self-evaluation of achievement on the Goal Attainment Scale; which skeletal muscles were injected; physiotherapists' and occupational therapists' evaluation of the patients' achievement of objectives on the Goal Attainment Scale; and proposals for optimization of treatment and changing goals......The aim of this study is to develop a treatment diary for patients receiving spasticity treatment including botulinum toxin injection and physiotherapy and/or occupational therapy. The diary focuses on problems triggered by skeletal muscle overactivity; agreed goals for treatment and the patient....... The evaluation included a satisfaction questionnaire and the WHO-QoL BREF and WHO-5 well-being score. Overall, 10 patients were enrolled in the pilot study. The patients were generally satisfied with the diary, found that it involved them more in their treatment and made it easier to set personal goals...

  12. Botulinum toxin drugs: brief history and outlook.

    Science.gov (United States)

    Dressler, D

    2016-03-01

    The global botulinum toxin (BT) market is currently undergoing rapid changes: this may be the time to review the history and the future of BT drug development. Since the early 1990s Botox(®) and Dysport(®) dominated the international BT market. Later, Myobloc(®)/NeuroBloc(®), a liquid BT type B drug, came out, but failed. Xeomin(®) is the latest major BT drug. It features removal of complexing proteins and improved neurotoxin purity. Several new BT drugs are coming out of Korea, China and Russia. Scientific challenges for BT drug development include modification of BT's duration of action, its transdermal transport and the design of BT hybrid drugs for specific target tissues. The increased competition will change the global BT market fundamentally and a re-organisation according to large indication groups, such as therapeutic and cosmetic applications, might occur.

  13. [Botulinum toxin and facial palsy. Our experience].

    Science.gov (United States)

    Navarrete Alvaro, María Luisa; Junyent, Josefina; Torrent, Luisa

    2010-01-01

    Therapeutic indication of peripheral facial paralysis depends on the degree of nerve injury. Severe facial palsy (electroneuronographic study less than or equal to 10%) leads to healing with sequelae. The sequelae of facial paralysis are contractures, hemifacial spasm and synkinesis.Our purpose was to demonstrate that these patients could benefit from rehabilitation treatment. We present a study of 48 patients with severe peripheral facial paralysis. They were treated from the beginning of reinnervation with botulinum toxin and facial exercises according to the Wisconsin School. The subjective efficacy of rehabilitation is high. Rehabilitation treatment can inform patients about their chances of recovery, give them control over and quality of facial expression and help to achieve greater facial symmetry. These factors provide better functionality and quality of life. Copyright 2009 Elsevier España, S.L. All rights reserved.

  14. Botulinum toxin A treatment of Raynaud's phenomenon: a review.

    Science.gov (United States)

    Iorio, Matthew L; Masden, Derek L; Higgins, James P

    2012-02-01

    Botulinum toxin A has conventionally been used in the upper extremity to treat spasticity resulting from stroke, paraplegia, and dystonia. Recently, it has been used to relieve symptoms of vasospasm in Raynaud's phenomenon. This review summarizes the current literature on botulinum toxin A in the treatment of Raynaud's phenomenon and examines the proposed mechanisms of action, suggested techniques of administration, and clinical efficacy. An Ovid MEDLINE search from 1950 to September 2010 was performed to identify any reports on the use of Botulinum toxin in the treatment of Raynaud's disease or associated vasoconstrictive disorders. All studies pertaining to "Raynaud's disease," "Raynaud's," or "vasoconstriction" were queried and meshed with a secondary search of studies pertaining to "botox" or "botulinum toxin type A." These reports were meshed and subsequently limited to human studies. All studies that met criteria were included and their outcomes evaluated and summarized. Since 2004, there have been 5 studies that have evaluated the use of Botulinum Toxin A for the treatment of Raynaud's. In each study, patients received a range of botulinum toxin injections (10-100 units) in their fingers and hands. The studies have many limitations (lack of controls, variable severity of disease, variability of dosing) but all report favorable clinical results. All showed overall improvement in patient pain as well as a reduction in soft tissue ulceration. Initial reports on the use of botulinum toxin A for Raynaud's phenomenon are promising. Larger controlled trials with improved study design are warranted. A better understanding of the mechanism of action, appropriate dose and dose frequency, and the efficacy relative to other medical and surgical treatments requires investigation. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Clostridium thermosaccharolyticum strain deficient in acetate production

    Energy Technology Data Exchange (ETDEWEB)

    Rothstein, D.M.

    1986-01-01

    A mutant of Clostridium thermosaccharolyticum that is blocked in acetate production was isolated after treatment with nitrosoguanidine and selection for fluoroacetate resistance. The mutant produced more ethanol than the parent strain did.

  16. Botulinum toxin for motor and phonic tics in Tourette's syndrome.

    Science.gov (United States)

    Pandey, Sanjay; Srivanitchapoom, Prachaya; Kirubakaran, Richard; Berman, Brian D

    2018-01-05

    Gilles de la Tourette syndrome, or Tourette's syndrome, is defined as the presence of both motor and vocal (phonic) tics for more than 12 months, that manifest before the age of 18 years, in the absence of secondary causes. Treatment of motor and phonic tics is difficult and challenging. To determine the safety and effectiveness of botulinum toxin in treating motor and phonic tics in people with Tourette's syndrome, and to analyse the effect of botulinum toxin on premonitory urge and sensory tics. We searched the Cochrane Movement Disorders Group Trials Register, CENTRAL, MEDLINE, and two trials registers to 25 October 2017. We reviewed reference lists of relevant articles for additional trials. We considered all randomised, controlled, double-blind studies comparing botulinum toxin to placebo or other medications for the treatment of motor and phonic tics in Tourette's syndrome for this review. We sought both parallel group and cross-over studies of children or adults, at any dose, and for any duration. We followed standard Cochrane methods to select studies, assess risk of bias, extract and analyse data. All authors independently abstracted data onto standardized forms; disagreements were resolved by mutual discussion. Only one randomised placebo-controlled, double-blind cross-over study met our selection criteria. In this study, 20 participants with motor tics were enrolled over a three-year recruitment period; 18 (14 of whom had a diagnosis of Tourette's syndrome) completed the study; in total, 21 focal motor tics were treated. Although we considered most bias domains to be at low risk of bias, the study recruited a small number of participants with relatively mild tics and provided limited data for our key outcomes. The effects of botulinum toxin injections on tic frequency, measured by videotape or rated subjectively, and on premonitory urge, are uncertain (very low-quality evidence). The quality of evidence for adverse events following botulinum toxin was

  17. Treatment of palatal myoclonus with botulinum toxin injection.

    Science.gov (United States)

    Anis, Mursalin M; Pollak, Natasha

    2013-01-01

    Palatal myoclonus is a rare cause of pulsatile tinnitus in patients presenting to the otolaryngology office. Rhythmic involuntary contractions of the palatal muscles produce the pulsatile tinnitus in these patients. Treatment of this benign but distressing condition with anxiolytics, anticonvulsants, and surgery has been largely unsuccessful. A few investigators have obtained promising results with botulinum toxin injection into the palatal muscles. We present a patient with palatal myoclonus who failed conservative treatment with anxiolytics. Unilateral injection of botulinum toxin into her tensor veli palatini muscle under electromyographic guidance resolved pulsatile tinnitus in her ipsilateral ear and unmasked pulsatile tinnitus in the contralateral ear. A novel method of following transient postinjection symptoms using a diary is presented in this study. Botulinum toxin dose must be titrated to achieve optimal results in each individual patient, analogous to titrations done for spasmodic dysphonia. Knowledge of the temporal onset of postinjection side effects and symptomatic relief may aid physicians in dose titration and surveillance. We present suggestions on titrating the botulinum toxin dose to optimal levels. A review of the literature on the use of botulinum toxin for palatal myoclonus and some common complications are discussed.

  18. Bupivacaine and botulinum toxin to treat comitant strabismus

    Directory of Open Access Journals (Sweden)

    Luisa Moreira Hopker

    2012-04-01

    Full Text Available PURPOSE: To evaluate the change in ocular motility and muscle thickness measured with ultrasonography after intramuscular injection of bupivacaine and botulinum toxin A. METHODS: Eight patients (five female were enrolled to measure ocular motility prior and 1, 7, 30 and 180 days after one injection of 2 ml of 1.5% bupivacaine and 2.5 U of botulinum toxin A in agonist and antagonist muscles, respectively, of eight amblyopic eyes. Muscle thickness was measured prior and on days 1, 7 and 30 after injection using 10-MHz ultrasonography (eyelid technique. RESULTS: Mean change in alignment was 10 prism diopters after 180 days (n=6. An average increase of 1.01 mm in muscle thickness was observed after 30 days of bupivacaine injection and 0.28 mm increase was observed after botulinum toxin A injection, as measured by ultrasonography. Lateral rectus muscles injected with bupivacaine had a mean increase of 1.5 mm in muscle thickness. CONCLUSION: In this study, a change in ocular motility was observed after 180 days of intramuscular injection of bupivacaine and botulinum toxin in horizontal extraocular muscles. Overall, there was an increase of muscle thickness in both botulinum toxinum A and bupivacaine injected muscles after 30 days of injection when measured by ultrasonography. This change was more pronounced on lateral rectus muscles after bupivacaine injection.

  19. TREATMENT OF LARYNGEAL DYSTONIA WITH BOTULINUM TOXIN: A CASE REPORT

    Directory of Open Access Journals (Sweden)

    Irena Hočevar-Boltežar

    2002-07-01

    Full Text Available Background. Laryngeal dystonia (LD is a form of focal dystonia, involving laryngeal muscles and causing disturbed phonation. Adductor, abductor and mixed forms of LD are described.Methods. Largely unsuccessful treatments include voice therapy, psychotherapy, biofeedback, section of the laryngeal recurrent nerve, laryngeal framework surgery and the use of centrally acting drugs. Botulinum toxin proved to be the most efficient treatment of LD; it acts by chemically denervating hyperactive laryngeal muscles.Case report. Authors report on the first use of botulinum toxin in a patient with LD in Slovenia. A female patient with a degenerative disease of extrapyramidal, cerebellar and autonomous system (probable multiple system atrophy and severe adductor form of LD is presented. By gradually increasing dose, the optimal dosage of botulinum has been established that greatly improves the patient’s speech for four months and causes transient mild adverse effects (dysphagia. Botulinum is injected percutaneously under endoscopic control, using the method, which has been described in literature for the first time.Conclusions. Treatment of LD with botulinum toxin demonstrated good results. It improves patient’s communication with others and the quality of his/her life.

  20. Type A botulinum neurotoxin complex proteins differentially modulate host response of neuronal cells.

    Science.gov (United States)

    Wang, Lei; Sun, Yi; Yang, Weiping; Lindo, Paul; Singh, Bal Ram

    2014-05-01

    Type A Botulinum neurotoxin (BoNT/A), the most potent poison known to mankind, is produced by Clostridium botulinum type A as a complex with neurotoxin-associated proteins (NAPs). Currently BoNT/A in purified and complex forms are both available in therapeutic and cosmetic applications to treat neuromuscular disorders. Whereas Xeomin(®) (incobotulinumtoxin A, Merz Pharmaceuticals, Germany) is free from complexing proteins, Botox(®) (onabotulinumtoxin A, Allergan, USA) contains NAPs, which by themselves have no known role in the intracellular biochemical process involved in the blockade of neurotransmitter release. Since the fate and possible interactions of NAPs with patient tissues after intramuscular injection are not known, it was the aim of this study to evaluate the binding of BoNT/A and/or the respective NAPs to cells derived from neuronal and non-neuronal human tissues, and to further explore neuronal cell responses to different components of BoNT/A. BoNT/A alone, the complete BoNT/A complex, and the NAPs alone, all bind to neuronal SH-SY5Y cells. The BoNT/A complex and NAPs additionally bind to RMS13 skeletal muscle cells, TIB-152 lymphoblasts, Detroit 551 fibroblasts besides the SH-SY5Y cells. However, no binding to these non-neuronal cells was observed with pure BoNT/A. Although BoNT/A, both in its purified and complex forms, bind to SH-SY5Y, the intracellular responses of the SH-SY5Y cells to these BoNT/A components are not clearly understood. Examination of inflammatory cytokine released from SH-SY5Y cells revealed that BoNT/A did not increase the release of inflammatory cytokines, whereas exposure to NAPs significantly increased release of IL-6, and MCP-1, and exposure to BoNT/A complex significantly increased release of IL-6, MCP-1, IL-8, TNF-α, and RANTES vs. control, suggesting that different components of BoNT/A complex induce significantly differential host response in human neuronal cells. Results suggest that host response to different

  1. Subcloning and Assessment of the Expression of Synthetic Gene of Botulinum Neurotoxin Type A Binding Domain (BD/A

    Directory of Open Access Journals (Sweden)

    Majid Zavvary

    2016-08-01

    Full Text Available Background and Objectives: Botulism syndrome is caused by Clostridium botulinum neurotoxin. This neurotoxin has seven serotypes ranging from A to G. The best way to prevent botulism syndrome caused by Botulinum neurotoxin (BoNT, is using recombinant vaccine made from its binding domain (due to having sufficient epitopes to stimulate immune system. In this study, the binding domain of BoNT serotype A (BoNT/A, was investigated. Methods: Initially, BoNT/A gene with accession number CP000727.1, was obtained from GenBank and was codon optimized according to the codon usage of E. coli. Then, the sequence was synthesized in pET28a plasmid and then subcloned in pGEX-4T-1 expression plasmid. The subcloning was done using PCR with Pfu DNA polymerase and then double digestion with XmaI and XhoI restriction enzymes. E. coli BL21 strain was used as the expression host. The selected marker for pGEX-4T-1 was ampicillin. Results: PCR and restriction digestion with the mentioned enzymes confirmed the subcloning process. The assessment of gene expression was performed by SDS-PAGE and western blotting (using horse anti-BoNT/A (and then glutathione affinity chromatography was performed. Although, the subcloning was performed successfully, no protein expression was observed.  Conclusion: According to the findings of this study, it seems that other hosts, such as eukaryotic hosts should be used for recombinant expression of BoNT/A binding domain.

  2. Role of neurotoxin associated proteins in the low pH induced structural changes in the botulinum neurotoxin complex.

    Science.gov (United States)

    Chellappan, Gowri; Kumar, Raj; Cai, Shuowei; Singh, Bal Ram

    2014-12-01

    Botulinum Neurotoxin (BoNT) produced by the bacterium Clostridium botulinum as a complex with NAPs causes botulism. It has been known that the NAPs protect the toxin from both extremes of pHs and proteases of the GI tract. In an attempt to emulate the physiological conditions encountered by the toxin, we examined BoNT/A, BoNT/A complex, and NAPs under different pH conditions and monitored their structural characteristics by far-UV CD and thermal denaturation analysis. BoNT/A complex showed the maximum CD signal with a mean residue weight ellipticity of -1.8 × 10(5)° cm(2)/dmol at 222 nm at both acidic and neutral pHs. Thermal denaturation analysis revealed NAPs to be the most stable amongst the three protein samples examined. Interestingly and quite uniquely, at pH 2.5, there was an increase in CD signal for BoNT complex as a function of temperature, which correlated with the NAPs profile, indicating a shielding effect of NAPs on BoNT complex at low pH. Calculation of the weighted mean of the ellipticities at the Tm for thermal unfolding of toxin and NAPs at neutral and acidic pHs showed variation with that of BoNT complex, suggesting structural reorganization in BoNT complex upon the association of NAPs and BoNT. In conclusion, this study reveals the structural behavior of BoNT complex and NAPs with pH changes substantially, which could be quite relevant for BoNT survival under extreme pH conditions in vivo.

  3. Analysis of active site residues of botulinum neurotoxin E by mutational, functional, and structural studies: Glu335Gln is an apoenzyme.

    Science.gov (United States)

    Agarwal, Rakhi; Binz, Thomas; Swaminathan, Subramanyam

    2005-06-14

    Clostridial neurotoxins comprising the seven serotypes of botulinum neurotoxins and tetanus neurotoxin are the most potent toxins known to humans. Their potency coupled with their specificity and selectivity underscores the importance in understanding their mechanism of action in order to develop a strategy for designing counter measures against them. To develop an effective vaccine against the toxin, it is imperative to achieve an inactive form of the protein which preserves the overall conformation and immunogenicity. Inactive mutants can be achieved either by targeting active site residues or by modifying the surface charges farther away from the active site. The latter affects the long-range forces such as electrostatic potentials in a subtle way without disturbing the structural integrity of the toxin causing some drastic changes in the activity/environment. Here we report structural and biochemical analysis on several mutations on Clostridium botulinum neurotoxin type E light chain with at least two producing dramatic effects: Glu335Gln causes the toxin to transform into a persistent apoenzyme devoid of zinc, and Tyr350Ala has no hydrolytic activity. The structural analysis of several mutants has led to a better understanding of the catalytic mechanism of this family of proteins. The residues forming the S1' subsite have been identified by comparing this structure with a thermolysin-inhibitor complex structure.

  4. Hazard analysis and possibilities for preventing botulism originating from meat products

    Directory of Open Access Journals (Sweden)

    Vasilev Dragan

    2008-01-01

    Full Text Available The paper presents the more important data on the bacteria Clostridium botulinum, the appearance of botulism, hazard analysis and the possibilities for preventing botulism. Proteolytic strains of C.botulinum Group I, whose spores are resistant to heat, create toxins predominantly in cans containing slightly sour food items, in the event that the spores are not inactivated in the course of sterilization. Non-proteolytic strains of Group II are more sensitive to high temperatures, but they have the ability to grow and create toxins at low temperatures. Type E most often creates a toxin in vacuum-packed smoked fish, and the non-proteolytic strain type B in dried hams and certain pasteurized meat products. The following plays an important role in the prevention of botulism: reducing to a minimum meat contamination with spores of clostridia, implementing good hygiene measures and production practice during the slaughter of animals, the inactivation of spores of C. botulinum during sterilization (F>3, and, in dried hams and pasteurized products, the prevention of bacterial growth and toxin forming by maintaining low temperatures in the course of production and storage, as well as the correct use of substances that inhibit the multiplication of bacteria and the production of toxins (nitrites, table salt, etc..

  5. Presence and molecular characterization of Clostridium difficile and Clostridium perfringens in intestinal compartments of healthy horses

    DEFF Research Database (Denmark)

    Schoster, Angelika; Arroyo, Luis Guillermo; Staempfli, Henry Rolf

    2012-01-01

    BACKGROUND: Clostridium difficile and Clostridium perfringens are commonly associated with colitis in equids, but healthy carriers exist. Scarce information is available on the prevalence of Clostridium spp. in gastrointestinal compartments other than faeces in healthy horses, and it is unknown...... colon and rectum. When multiple compartments were positive in a single horse, two different C. difficile ribotypes were always present. Clostridium perfringens Type A (CPE, beta2 toxin gene negative) was recovered from the left ventral colon of one horse (0.74%, 1/135 samples). Agreement between faeces...... and overall C. difficile carrier status was good. CONCLUSIONS: Clostridium difficile can be found in different compartments of the gastrointestinal tract of healthy horses, and multiple strains can be present in an individual horse. The prevalence of C. perfringens in healthy adult hoses was low, consistent...

  6. Toxinas de Clostridium perfringens Toxins of Clostridium perfringens

    Directory of Open Access Journals (Sweden)

    W. E. Morris

    2009-12-01

    Full Text Available Clostridium perfringens es un bacilo grampositivo anaerobio con capacidad de formar esporas. Es uno de los patógenos bacterianos con mayor distribución en el medio ambiente, ya que puede ser aislado de muestras de suelo y de agua y además forma parte de la microbiota intestinal de animales y humanos. Sin embargo, en ciertas ocasiones puede actuar como patógeno oportunista y causar enfermedades como la gangrena gaseosa, la enterotoxemia del ovino y del caprino y la disentería del cordero, entre otras. En humanos, está asociado a enfermedades como la intoxicación por alimentos, la enterocolitis necrotizante en niños y la enteritis necrótica o pigbel de las tribus de Papúa-Nueva Guinea. El renovado interés que existe actualmente en el estudio de C. perfringens como patógeno veterinario y humano, junto con el avance de la biología molecular, han hecho posible que la ciencia tenga hoy un conocimiento más profundo sobre la biología y la patogenia de esta bacteria. En esta revisión bibliográfica se discuten y actualizan los principales aspectos de la patogenia intestinal de C. perfringens teniendo en cuenta las toxinas con mayor importancia médica descritas hasta el presente.Clostridium perfringens is an anaerobic gram-positive spore-forming bacillus. It is one of the pathogens with larger distribution in the environment; it can be isolated from soil and water samples, which also belongs to the intestinal flora of animals and humans. However, on some occasions it can act as an opportunistic pathogen, causing diseases such as gas gangrene, enterotoxemia in sheep and goats and lamb dysentery, among others. In human beings, it is associated to diseases such as food poisoning, necrotic enterocolitis of the infant and necrotic enteritis or pigbel in Papua-New Guinea tribes. The renewed interest existing nowadays in the study of C. perfringens as a veterinarian and human pathogen, together with the advance of molecular biology, had enabled

  7. Botulinum toxin treatment of lower extremity spasticity

    Directory of Open Access Journals (Sweden)

    S. E. Khat’kova

    2017-01-01

    Full Text Available The article reviews the current concept of lower extremity spasticity, which is a frequent disabling consequence of stroke. Gait biomechanics, step cycle and main pathologic patterns of lower extremity are described (hip adduction, knee flexion, knee extension, foot plantar flexion, equinovarus foot position, toes flexion, hallux extension, including muscles involved in the pathological process. Additionally the article contains detailed information on pathologic principles of lower extremity spasticity development. Special focus is given to sarcomeregenesis as an essential element of the development of potential conditions for muscle tissue adaptation to a new state and restoration of muscle length and strength. At present Botulinum toxin A (BTA is used in a complex spasticity management programs. The results of clinical studies performed in the last decade supporting the efficacy of Botox® (Onabotulinumtoxin A in the treatment of spasticity are reviewed. Effective BTA doses are proposed. Authors came to the conclusion that BTA as a part of complex rehabilitation in patients with poststroke spasticity of lower extremity promotes treatment efficacy due to a decrease of muscle tone and increase of range of movements in the joints. BTA should be regarded as an essential part of standard rehabilitation programs. Further studies to define optimal muscles for intervention, BTA doses and rehabilitation schemes are still needed. 

  8. Why do females use botulinum toxin injections?

    Directory of Open Access Journals (Sweden)

    Carter Singh

    2015-01-01

    Full Text Available Background: Botulinum toxin (BT use for enhancing the facial features has become a commonly accepted form of aesthetic intervention. This study conducted a self-report survey of female BT users in order to explore the motivating factors in its use (cost-benefit analysis. Settings and Design: This is a cross-sectional exploratory pilot study. Materials and Methods: Self-report questionnaires were administered to 41 consecutive clients attending an independent medical practice for BT injections for cosmetic purposes. All the participants were females and represented a range of age groups from the 20s to above 60s. Items in the nonstandardized questionnaire elicited questions relating to the reasons for and against BT use. Statistical Analysis Used: Descriptive analysis was used rather than inferential statistics, and involved ranking the responses according to the most likely reasons for using BT and disadvantages of its use. Results: In general, the primary motivating factor for BT use was to improve self-esteem, and the greatest disadvantage involved financial costs associated with the procedure. Conclusions: The main findings of this study suggest that females who use BT for aesthetic purposes are motivated by personal psychological gains (intrapersonal attributes rather than social gains (interpersonal factors. In other words, they do not believe that having BT will equate to being treated any better by other people but would rather provide them with confidence and satisfaction regarding their self-image.

  9. Botulinum neurotoxin: a marvel of protein design.

    Science.gov (United States)

    Montal, Mauricio

    2010-01-01

    Botulinum neurotoxin (BoNT), the causative agent of botulism, is acknowledged to be the most poisonous protein known. BoNT proteases disable synaptic vesicle exocytosis by cleaving their cytosolic SNARE (soluble NSF attachment protein receptor) substrates. BoNT is a modular nanomachine: an N-terminal Zn(2+)-metalloprotease, which cleaves the SNAREs; a central helical protein-conducting channel, which chaperones the protease across endosomes; and a C-terminal receptor-binding module, consisting of two subdomains that determine target specificity by binding to a ganglioside and a protein receptor on the cell surface and triggering endocytosis. For BoNT, functional complexity emerges from its modular design and the tight interplay between its component modules--a partnership with consequences that surpass the simple sum of the individual component's action. BoNTs exploit this design at each step of the intoxication process, thereby achieving an exquisite toxicity. This review summarizes current knowledge on the structure of individual modules and presents mechanistic insights into how this protein machine evolved to this level of sophistication. Understanding the design principles underpinning the function of such a dynamic modular protein remains a challenging task.

  10. Treatment of Chronic Migraine with Focus on Botulinum Neurotoxins

    Directory of Open Access Journals (Sweden)

    Sara M. Schaefer

    2015-07-01

    Full Text Available Migraine is the most common neurological disorder, and contributes to disability and large healthcare costs in the United States and the world. The treatment of migraine until recently has focused on medications, both abortive and prophylactic, but treatment of chronic migraine has been revolutionized with the introduction of botulinum toxin injection therapy. In this review, we explore the current understanding of migraine pathophysiology, and the evolution of the use of botulinum toxin therapy including proposed pathophysiological mechanisms through animal data. We also discuss the similarities and differences between three injection techniques.

  11. The Zinc-Dependent Protease Activity of the Botulinum Neurotoxins

    Science.gov (United States)

    Lebeda, Frank J.; Cer, Regina Z.; Mudunuri, Uma; Stephens, Robert; Singh, Bal Ram; Adler, Michael

    2010-01-01

    The botulinum neurotoxins (BoNT, serotypes A-G) are some of the most toxic proteins known and are the causative agents of botulism. Following exposure, the neurotoxin binds and enters peripheral cholinergic nerve endings and specifically and selectively cleaves one or more SNARE proteins to produce flaccid paralysis. This review centers on the kinetics of the Zn-dependent proteolytic activities of these neurotoxins, and briefly describes inhibitors, activators and factors underlying persistence of toxin action. Some of the structural, enzymatic and inhibitor data that are discussed here are available at the botulinum neurotoxin resource, BotDB (http://botdb.abcc.ncifcrf.gov). PMID:22069621

  12. In-vitro growth characteristics of commercial probiotic strains and their potential for inhibition of Clostridium difficile and Clostridium perfringens

    DEFF Research Database (Denmark)

    Schoster, A.; Kokotovic, Branko; Permin, A.

    2012-01-01

    Probiotics have gained importance in human and veterinary medicine to prevent enteric disease. Little information is available on commercial probiotic strains regarding their growth characteristics and inhibition of equine enteric pathogens such as Clostridium difficile and Clostridium perfringens...

  13. Behaviour of the pathogen surrogates Listeria innocua and Clostridium sporogenes during production of parsley in fields fertilized with contaminated amendments.

    Science.gov (United States)

    Girardin, Hélène; Morris, Cindy E; Albagnac, Christine; Dreux, Nicolas; Glaux, Catherine; Nguyen-The, Christophe

    2005-10-01

    The survival and transfer of Listeria innocua and Clostridium sporogenes, used as surrogates of the food borne pathogens Listeria monocytogenes and Clostridium botulinum, were quantitatively assessed under field conditions. In the soil, spores of C. sporogenes declined by less than 0.7 log cycles within 16 months and were detected on parsley leaves throughout the experiment. In contrast, L. innocua in the soil declined by 7 log cycles in 90 days and was detected on leaves in low numbers (>0.04 MPN g(-1)) during the first 30 days. Rates of decline in soil were similar in the laboratory at 20 degrees C for two strains of L. innocua and L. monocytogenes ; and in the field for L. innocua over two different years. L. innocua survived better in winter, indicating an important influence of temperature. The major cause of transfer of L. innocua from soil to parsley leaves was splashing due to rain and irrigation. As few as 1 CFU g(-1) Listeria in soil led to contamination of parsley leaves. Internalisation of Listeria through parsley roots was not observed. Under the conditions of soil and climate studied, a delay of 90 days between application of potentially contaminated fertilizer and harvest should be sufficient to eliminate L. monocytogenes.

  14. Adjuvants for Clostridium tetani and Clostridium diphtheriae vaccines updating.

    Science.gov (United States)

    Alshanqiti, Fatimah M; Al-Masaudi, Saad B; Al-Hejin, Ahmed M; Redwan, Elrashdy M

    2017-01-01

    It's known that diphtheria and tetanus are a contagious lethal diseases over the years, they caused by pathogenic microbes corynebacterium diphtheria and Clostridium tetani, respectively. The diseases result from the production of bacterial toxin. Vaccination with bacterial toxoid vaccines adsorbed on particulates adjuvants still are the best way to prevent this epidemic diseases from spread. The particulate vaccines have been shown to be more efficient than soluble one for the induction of the immune responses. Nanoparticles can be engineered to enhance the immune responses. As well known the immune response to inactivate killed and subunit vaccine enhances by alum adjuvants. The adjuvants examined and tested after reducing its size to particle size, thus mimic size of viruses which is considered smallest units can derive the immune system. The major issue is minimizing the adjuvant particles, to gain insight of resulting immunity types and impact on immune response. The adjuvant effect of micro/nanoparticles appears to largely be a consequence of their uptake into antigen presenting cells.

  15. Solubility of the catalytic domains of Botulinum neurotoxin serotype E subtypes.

    Science.gov (United States)

    Chen, Sheng; Barbieri, Joseph T

    2016-02-01

    The Clostridium botulinum neurotoxins (BoNTs) are the most potent protein toxins known to humans. There are seven serotypes of the BoNTs (A-G), among which serotypes A, B, E and F are known to cause natural human intoxication. To date, eleven subtypes of LC/E, termed E1∼E11, have been identified. The LCs of BoNT/E were insoluble, prohibiting studies towards understanding the mechanisms of toxin action and substrate recognition. In this work, the molecular basis of insolubility of the recombinant LCs of two representative subtypes of BoNT/E, E1(Beluga) and E3 (Alaska), was determined. Hydrophobicity profile and structural modeling predicted a C-terminal candidate region responsible for the insolubility of LC/Es. Deletion of C-terminal 19 residues of LC/E(1-400) resulted in enhanced solubility, from 2 to ∼50% for LC/EAlaska and from 16 to ∼95% for LC/EBeluga. In addition, resides 230-236 were found to contribute to a different solubility level of LC/EAlaska when compared to LC/EBeluga. Substituting residues (230)TCI(232) in LC/EAlaska to the corresponding residues of (230)KYT(232) in LC/EBeluga enhanced the solubility of LC/EAlaska to a level approaching that of LC/EBeluga. Among these LC/Es and their derivatives, LC/EBeluga 1-400 was the most soluble and stable protein. Each LC/E derivative possessed similar catalytic activity, suggesting that the C-terminal region of LC/Es contributed to protein solubility, but not catalytic activity. In conclusion, this study generated a soluble and stable recombinant LC/E and provided insight into the structural components that govern the solubility and stability of the LCs of other BoNT serotypes and Tetanus toxin. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Polyclonal Antibody Therapies for Clostridium difficile Infection

    Directory of Open Access Journals (Sweden)

    Michael R. Simon

    2014-10-01

    Full Text Available Clostridium difficile infection has emerged as a growing worldwide health problem. The colitis of Clostridium difficile infection results from the synergistic action of C. difficile secreted toxins A and B upon the colon mucosa. A human monoclonal IgG anti-toxin has demonstrated the ability in combination therapy to reduce mortality in C. difficile challenged hamsters. This antibody is currently in a clinical trial for the treatment of human Clostridium difficile infection. More than one group of investigators has considered using polyclonal bovine colostral antibodies to toxins A and B as an oral passive immunization. A significant proportion of the healthy human population possesses polyclonal antibodies to the Clostridium difficile toxins. We have demonstrated that polyclonal IgA derived from the pooled plasma of healthy donors possesses specificity to toxins A and B and can neutralize these toxins in a cell-based assay. This suggests that secretory IgA prepared from such pooled plasma IgA may be able to be used as an oral treatment for Clostridium difficile infection.

  17. Use of Botulinum Neurotoxin Injections to Treat Spasticity

    Science.gov (United States)

    ... your family understand the use of BoNT for treatment of spasticity, a disorder following injury to the brain or ... available evidence on the effect of BoNT for treatment of spasticity. What is botulinum neurotoxin and how does it ...

  18. Electromyography in cervical dystonia: changes after botulinum and trihexyphenidyl

    NARCIS (Netherlands)

    Brans, J. W.; Aramideh, M.; Koelman, J. H.; Lindeboom, R.; Speelman, J. D.; Ongerboer de Visser, B. W.

    1998-01-01

    BACKGROUND: The value of physical examination in detecting involved neck muscles in cervical dystonia (CD) is uncertain and little is known about changes in electromyographic (EMG) features after botulinum toxin type A (BTA) treatment. METHODS: In a double-blind, randomized study we recorded the EMG

  19. Botulinum toxin in cervical dystonia: low dosage with electromyographic guidance

    NARCIS (Netherlands)

    Brans, J. W.; de Boer, I. P.; Aramideh, M.; Ongerboer de Visser, B. W.; Speelman, J. D.

    1995-01-01

    Sixty patients with idiopathic cervical dystonia were treated a total of 240 times with botulinum toxin type A (BTA). Selected muscles were injected with BTA under electromyographic (EMG) guidance. The clinical effect was measured on the Tsui scale and a 10-point anchored visual analogue scale. A

  20. Pretarsal application of botulinum toxin for treatment of blepharospasm

    NARCIS (Netherlands)

    Aramideh, M.; Ongerboer de Visser, B. W.; Brans, J. W.; Koelman, J. H.; Speelman, J. D.

    1995-01-01

    The response to botulinum toxin type A was compared after two injection techniques in 45 patients with blepharospasm. Initially, patients were treated according to a triple injection technique; two injections into the upper eyelid and one injection into the lower eyelid. Subsequently, without

  1. Does motor performance matter in botulinum toxin efficacy for drooling?

    NARCIS (Netherlands)

    Erasmus, C.E.; Scheffer, A.R.T.; Hulst, K. van; Limbeek, J. van; Hoogen, F.J.A. van den; Rotteveel, J.J.; Jongerius, P.H.

    2011-01-01

    The aim of this study was to define factors that influence therapy outcome of submandibular botulinum toxin injections for drooling in children with cerebral palsy or mental disability. We postulated that differences in response may be explained by the variation of dysfunctions in the various

  2. [Treatment of severe bruxism with botulinum toxin type A].

    Science.gov (United States)

    Alonso-Navarro, Hortensia; Jiménez-Jiménez, Félix J; Plaza-Nieto, José F; Pilo-De la Fuente, Belén; Navacerrada, Francisco; Arroyo-Solera, Margarita; Calleja, Marisol

    2011-07-16

    The possible usefulness of botulinum toxin type A in the treatment of bruxism has not been studied exhaustively, being limited to some isolated case reports, two short case-series and a double-blind study involving a small number or patients. This article report our long-term experience in the treatment of bruxism with botulinum toxin type A. The outcome of 19 patients with severe bruxism who underwent periodical treatment with botulinum toxin A infiltrations in both temporal and masseter muscles, using initial doses of 25 IU per muscle, during a follow-up period ranging from 0.5 to 11 years, is described. Doses were adjusted in follow-up visits according the response degree. None of the patients reported side-effects. Final doses ranged from 25 to 40 IU per muscle (mean: 29.7 ± 4.9 UI), and duration of the effect from 13 to 26 weeks (mean: 16.7 ± 5.1 weeks). Botulinum toxin A infiltrations are a safe and useful treatment for patients with severe bruxism.

  3. Intramural injection with botulinum toxin significantly elongates the pig esophagus

    DEFF Research Database (Denmark)

    Larsen, Heidi Fhær; Jensen, Thorbjørn Søren Rønn; Rasmussen, Lars

    2013-01-01

    Surgical treatment of long-gap esophageal atresia (LGEA) is challenging. Methods which facilitate stretching of the esophageal pouches may allow primary anastomosis. Botulinum toxin type A (BTX-A) blocks acetylcholine release in neuromuscular junctions, thereby causing muscle relaxation. We...

  4. Experience with botulinum toxin in the treatment of cerebral palsy

    African Journals Online (AJOL)

    Fifteen children with cerebral palsy: 5 with dynamic spasticity, 5 with dystonia and 5 with a mixed picture. Results. On a standard scoring system, 13 of the children showed improved function at reassessment. Conclusion. Intramuscular injection of botulinum toxin is effective in the treatment of selected children with spastic.

  5. Botulinum Toxin – Treatment Of Choice For Primary Hyperhidrosis ...

    African Journals Online (AJOL)

    The term Botulinum toxin encompasses a group of seven (A – G) immunologically distinct Zinc-dependent endoproteases that act primarily at peripheral cholinergic synapses. By blocking synaptic vesicle trafficking they effectively block the release of Acetylcholine inducing muscle paralysis. This property is utilized in the ...

  6. Calf muscle volume estimates: Implications for Botulinum toxin treatment?

    DEFF Research Database (Denmark)

    Bandholm, Thomas; Sonne-Holm, Stig; Thomsen, Carsten

    2007-01-01

    An optimal botulinum toxin dose may be related to the volume of the targeted muscle. We investigated the suitability of using ultrasound and anthropometry to estimate gastrocnemius and soleus muscle volume. Gastrocnemius and soleus muscle thickness was measured in 11 cadaveric human legs, using...... the volume of individual plantar flexor muscles using ultrasound and anthropometry. This possibility should be investigated further in living humans....

  7. ISOLATION OF CLOSTRIDIUM TETANI FROM SOIL.

    Science.gov (United States)

    SANADA, I; NISHIDA, S

    1965-03-01

    Sanada, Ichiro (Kanazawa University, Kanazawa, Japan), and Shoki Nishida. Isolation of Clostridium tetani from soil. J. Bacteriol. 89:626-629. 1965.-The higher the temperatures applied to soil specimens, the weaker the toxigenicity of Clostridium tetani strains isolated from them. The glucose- and maltose-fermenting ability of these isolates was inversely proportional to their toxigenicity. The biological properties of atoxic strains were indistinguishable from those of C. tetanomorphum. Since a considerable number of toxic strains fermented glucose and maltose, these criteria are of doubtful value for differentiating C. tetani from C. tetanomorphum.

  8. Guidelines on the use of botulinum toxin Type A

    Directory of Open Access Journals (Sweden)

    Shetty M

    2008-03-01

    Full Text Available Botulinum toxin is available as types A and B. These two different forms need different dosages and hence, the physician needs to be familiar with the formulations. A thorough knowledge of the anatomy and physiology of the muscles in the area to be injected is essential. Indications for botulinum toxin: Dynamic wrinkles caused by persistent muscular contractions are the main aesthetic indications for the use of Botulinum toxin. These include forehead lines, glabellar lines, crow′s feet, bunny lines, perioral wrinkles, and platysmal bands. Non-aesthetic indications include hyperhidrosis of the palms, soles and axillae. Physicians′ qualifications : Any qualified dermatologist may practice the technique after receiving adequate training in the field. This may be obtained either during post-graduation or at any workshops dedicated to this subject. Facility: Botulinum toxin can be administered in the dermatologist′s minor procedure room. Preoperative counseling and informed consent Detailed counseling with respect to the treatment, desired effects, and longevity of the results should be discussed with the patient. The patient should be given brochures to study and adequate opportunity to seek information. A detailed consent form needs to be completed by the patient. The consent form should include the type of botulinum toxin, longevity expected and possible postoperative complications. Pre- and postoperative photography is recommended. Dosage depends on the area, muscle mass, gender and other factors outlined in these guidelines. It is recommended that beginners should focus on the basic indications in the upper third of the face and that they treat the middle and lower parts of the face only after garnering adequate experience.

  9. Effect of Botulinum Toxin and Surgery among Spasmodic Dysphonia Patients.

    Science.gov (United States)

    van Esch, Babette F; Wegner, Inge; Stegeman, Inge; Grolman, Wilko

    2017-02-01

    Objective The effect of botulinum toxin among patients with adductor spasmodic dysphonia (AdSD) is temporary. To optimize long-term treatment outcome, other therapy options should be evaluated. Alternative treatment options for AdSD comprise several surgical treatments, such as thyroarytenoid myotomy, thyroplasty, selective laryngeal adductor denervation-reinnervation, laryngeal nerve crush, and recurrent laryngeal nerve resection. Here, we present the first systematic review comparing the effect of botulinum toxin with surgical treatment among patients diagnosed with AdSD. Data Sources MEDLINE (PubMed), EMBASE, and the Cochrane Library. Methods Articles were reviewed by 2 independent authors, and data were compiled in tables for analysis of the objective outcome (voice expert evaluation after voice recording), the subjective outcome (patient self-assessment scores), and voice-related quality of life (Voice Health Index scores). Results No clinical trials comparing both treatment modalities were identified. Single-armed studies evaluated either the effect of botulinum toxin or surgical treatment. Thirteen studies reported outcomes after botulinum toxin treatment (n = 419), and 9 studies reported outcomes after surgical treatment (n = 585 patients). A positive effect of bilateral botulinum toxin injections was found for the objective voice outcome, subjective voice outcome, and quality of life. The duration of the beneficial effect ranged from 15 to 18 weeks. Surgical treatment had an overall positive effect on objective voice improvement, subjective voice improvement, and quality of live. Conclusion No preference for one treatment could be demonstrated. Prospective clinical trials comparing treatment modalities are recommended to delineate the optimal outcomes by direct comparison.

  10. Botulinum toxin type a for dysthyroid upper eyelid retraction.

    Science.gov (United States)

    Morgenstern, K E; Evanchan, J; Foster, J A; Cahill, K V; Burns, J A; Holck, D E E; Perry, J D; Wulc, A E

    2004-05-01

    To evaluate the safety and efficacy of botulinum toxin type A for treatment of eyelid retraction resulting from thyroid eye disease (TED) during the inflammatory phase of the condition. In this prospective, nonrandomized case series, 18 patients with inflammatory eyelid retraction caused by active TED received botulinum toxin type A injection (10, 5, or 2.5 U) for treatment of upper eyelid retraction. Botulinum toxin type A (Allergan, Irvine, CA, U.S.A.) was injected transconjunctivally just above the superior tarsal border in the elevator complex of the upper eyelid. Seventeen of 18 patients (94%) demonstrated a reduced marginal reflex distance (MRD1) after botulinum toxin injection. The average change in MRD1 of the treated eyelid after injection was -2.35 mm (range, 0 to -8.0 mm). Of the 27 eyelids injected, 33% had a 0- to 1-mm drop in eyelid height, 30% had a 1.5- to 2-mm decrease, 22% had a 2.5- to 3-mm decrease, and 15% had a greater than 3-mm decrease in eyelid height. None of the treated eyelids were noted to increase in height. One patient showed no alteration inafter treatment. One patient had clinically MRD1 significant ptosis and one patient reported worsening of preexisting diplopia after injection. Three patients undergoing unilateral injection had relative contralateral eyelid elevation. All untoward effects resolved spontaneously without sequelae. : Botulinum toxin type A may be used in the inflammatory stage of thyroid eye disease to improve upper eyelid retraction. Individual response to treatment is variable, but this modality should be considered as a temporizing measure until stability for surgery is reached.

  11. Subcloning and Assessment of the Expression of Synthetic Botulinum Neurotoxin Type B Binding Domain (BD/B

    Directory of Open Access Journals (Sweden)

    Majid Baradaran

    2016-07-01

    Full Text Available Background and Objectives: Botulism syndrome is caused by Clostridium botulinum neurotoxin. This neurotoxin has 7 serotypes, from A to G. The best way to prevent botulism syndrome caused by BoNT/B is immunization with recombinant binding domains of BoNT/B (due to containing sufficient epitopes to stimulate the immune system. In this study, the expression of the BoNT/B binding domain as a candidate vaccine, was investigated. Methods: At first, the sequence of the BoNT/B binding domain gene was obtained from NCBI website with accession number of EF028399.1. After codon optimization, the gene was synthesized in pUC18 vector. Then, the gene was subcloned in pET32a(+ expression vector that carries an ampicillin selection marker. PCR, enzymatic digestion, and sequencing were used to confirm subcloning accuracy, and E. coli BL21(DE3 was used for the expression analysis. The accuracy of protein expression was evaluated by electrophoresis and western blotting. Results: PCR reaction, enzymatic digestion, and sequencing confirmed that the gene of interest has been subcloned appropriately in the vector. The expression analysis by SDS-PAGE and subsequently western blotting, showed that the protein of interest is not expressed in the mentioned expression strain. Conclusion: Although the gene was successfully subcloned in pET32a(+ vector, no significant expression of the gene was observed.

  12. Botulinum Toxin B Affects Neuropathic Pain but Not Functional Recovery after Peripheral Nerve Injury in a Mouse Model

    Directory of Open Access Journals (Sweden)

    Alba Finocchiaro

    2018-03-01

    Full Text Available Clinical use of neurotoxins from Clostridium botulinum is well established and is continuously expanding, including in treatment of pain conditions. Background: The serotype A (BoNT/A has been widely investigated, and current data demonstrate that it induces analgesia and modulates nociceptive processing initiated by inflammation or nerve injury. Given that data concerning the serotype B (BoNT/B are limited, the aim of the present study was to verify if also BoNT/B is able not only to counteract neuropathic pain, but also to interfere with inflammatory and regenerative processes associated with the nerve injury. Methods: As model of neuropathic pain, chronic constriction injury (CCI of the sciatic nerve was performed in CD1 male mice. Mice were intraplantarly injected with saline (control or BoNT/B (5 or 7.5 pg/mouse into the injured hindpaw. For comparison, another mouse group was injected with BoNT/A (15 pg/mouse. Mechanical allodynia and functional recovery of the injured paw was followed for 101 days. Spinal cords and sciatic nerves were collected at day 7 for immunohistochemistry. Results and Conclusions: The results of this study show that BoNT/B is a powerful biological molecule that, similarly to BoNT/A, can reduce neuropathic pain over a long period of time. However, the analgesic effects are not associated with an improvement in functional recovery, clearly highlighting an important difference between the two serotypes for the treatment of this chronic pain state.

  13. Botulinum Toxin B Affects Neuropathic Pain but Not Functional Recovery after Peripheral Nerve Injury in a Mouse Model

    Science.gov (United States)

    Finocchiaro, Alba; Marinelli, Sara; De Angelis, Federica; Vacca, Valentina; Pavone, Flaminia

    2018-01-01

    Clinical use of neurotoxins from Clostridium botulinum is well established and is continuously expanding, including in treatment of pain conditions. Background: The serotype A (BoNT/A) has been widely investigated, and current data demonstrate that it induces analgesia and modulates nociceptive processing initiated by inflammation or nerve injury. Given that data concerning the serotype B (BoNT/B) are limited, the aim of the present study was to verify if also BoNT/B is able not only to counteract neuropathic pain, but also to interfere with inflammatory and regenerative processes associated with the nerve injury. Methods: As model of neuropathic pain, chronic constriction injury (CCI) of the sciatic nerve was performed in CD1 male mice. Mice were intraplantarly injected with saline (control) or BoNT/B (5 or 7.5 pg/mouse) into the injured hindpaw. For comparison, another mouse group was injected with BoNT/A (15 pg/mouse). Mechanical allodynia and functional recovery of the injured paw was followed for 101 days. Spinal cords and sciatic nerves were collected at day 7 for immunohistochemistry. Results and Conclusions: The results of this study show that BoNT/B is a powerful biological molecule that, similarly to BoNT/A, can reduce neuropathic pain over a long period of time. However, the analgesic effects are not associated with an improvement in functional recovery, clearly highlighting an important difference between the two serotypes for the treatment of this chronic pain state. PMID:29562640

  14. Clostridium difficile phages: still difficult?

    Directory of Open Access Journals (Sweden)

    Katherine Rose Hargreaves

    2014-04-01

    Full Text Available Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however developing suitable phages is challenging. In this review we summarise the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics.Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage-host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution.No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using whole-phages are such that they remain a major research focus. Whilst we don’t envisage working with C. difficile phages will be problem free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen.

  15. Clostridium difficile infection in Thailand.

    Science.gov (United States)

    Putsathit, Papanin; Kiratisin, Pattarachai; Ngamwongsatit, Puriya; Riley, Thomas V

    2015-01-01

    Clostridium difficile is the aetiological agent in ca. 20% of cases of antimicrobial-associated diarrhoea in hospitalised adults. Diseases caused by this organism range from mild diarrhoea to occasional fatal pseudomembranous colitis. The epidemiology of C. difficile infection (CDI) has changed notably in the past decade, following epidemics in the early 2000s of PCR ribotype (RT) 027 infection in North America and Europe, where there was an increase in disease severity and mortality. Another major event has been the emergence of RT 078, initially as the predominant ribotype in production animals in the USA and Europe, and then in humans in Europe. Although there have been numerous investigations of the epidemiology of CDI in North America and Europe, limited studies have been undertaken elsewhere, particularly in Asia. Antimicrobial exposure remains the major risk factor for CDI. Given the high prevalence of indiscriminate and inappropriate use of antimicrobials in Asia, it is conceivable that CDI is relatively common among humans and animals. This review describes the level of knowledge in Thailand regarding C. difficile detection methods, prevalence and antimicrobial susceptibility profile, as well as the clinical features of, treatment options for and outcomes of the disease. In addition, antimicrobial usage in livestock in Thailand will be reviewed. A literature search yielded 18 studies mentioning C. difficile in Thailand, a greater number than from any other Asian country. It is possible that the situation in Thailand in relation to CDI may mirror the situation in other developing Asians countries. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  16. Atypical Glycolysis in Clostridium thermocellum

    Science.gov (United States)

    Zhou, Jilai; Olson, Daniel G.; Argyros, D. Aaron; Deng, Yu; van Gulik, Walter M.; van Dijken, Johannes P.

    2013-01-01

    Cofactor specificities of glycolytic enzymes in Clostridium thermocellum were studied with cellobiose-grown cells from batch cultures. Intracellular glucose was phosphorylated by glucokinase using GTP rather than ATP. Although phosphofructokinase typically uses ATP as a phosphoryl donor, we found only pyrophosphate (PPi)-linked activity. Phosphoglycerate kinase used both GDP and ADP as phosphoryl acceptors. In agreement with the absence of a pyruvate kinase sequence in the C. thermocellum genome, no activity of this enzyme could be detected. Also, the annotated pyruvate phosphate dikinase (ppdk) is not crucial for the generation of pyruvate from phosphoenolpyruvate (PEP), as deletion of the ppdk gene did not substantially change cellobiose fermentation. Instead pyruvate formation is likely to proceed via a malate shunt with GDP-linked PEP carboxykinase, NADH-linked malate dehydrogenase, and NADP-linked malic enzyme. High activities of these enzymes were detected in extracts of cellobiose-grown cells. Our results thus show that GTP is consumed while both GTP and ATP are produced in glycolysis of C. thermocellum. The requirement for PPi in this pathway can be satisfied only to a small extent by biosynthetic reactions, in contrast to what is generally assumed for a PPi-dependent glycolysis in anaerobic heterotrophs. Metabolic network analysis showed that most of the required PPi must be generated via ATP or GTP hydrolysis exclusive of that which happens during biosynthesis. Experimental proof for the necessity of an alternative mechanism of PPi generation was obtained by studying the glycolysis in washed-cell suspensions in which biosynthesis was absent. Under these conditions, cells still fermented cellobiose to ethanol. PMID:23435896

  17. Description of Clostridium phoceensis sp. nov., a new species within the genus Clostridium

    Directory of Open Access Journals (Sweden)

    M. Hosny

    2016-11-01

    Full Text Available Clostridium phoceensis sp. nov., strain GD3T (= CSUR P1929 = DSM 100334 is the type strain of C. phoceensis sp. nov., a new species within the genus Clostridium. This strain was isolated from the gut microbiota of a 28-year-old healthy French man. C. phoceensis is a Gram-negative, spore-forming, nonmotile, strictly anaerobic bacterium. We describe its complete genome sequence and annotation, together with its phenotypic characteristics.

  18. RESISTANCE OF GUINEA PIGS IMMUNIZED WITH BOTULINUM TOXOIDS TO AEROGENIC CHALLENGE WITH TOXIN

    Science.gov (United States)

    guinea pigs with botulinum toxoids afforded a high level of protection to challenge with botulinum toxins by the respiratory route. Resistance to challenge by this route was similar to resistance afforded to challenge by the parenteral and oral routes. The high survival percentages obtained in these studies made it impossible to establish any relationship between serum antitoxin titer and resistance to challenge. This report presents results of some studies to determine resistance afforded actively immunized guinea pigs to challenge with botulinum

  19. Relationship of bacteriophages to alpha toxin production in Clostridium novyi types A and B.

    Science.gov (United States)

    Eklund, M W; Poysky, F T; Peterson, M E; Meyers, J A

    1976-09-01

    The relationship of specific bacteriophages to the production of the lethal alpha toxin in Clostridium novyi types A and B was investigated. When type A strain 5771 reverted to the phage-sensitive state, it ceased to produce alpha toxin but continued to produce the gamma and epsilon antigens. This "nontoxigenic" culture, therefore, more closely resembled C. botulinum types C and D than the other C. novyi types. Phage-sensitive type B strains also ceased to produce the alpha toxin but continued to produce the beta toxin, and therefore very colesly resembled C. novyi type D (C. haemolyticum). Alpha toxin was again produced when the phage-sensitive cultures were reinfected with the respective tox+ phages. Alpha toxin production could also be induced in the "nontoxigenic" phage-sensitive derivatives from type B strain 8024 by tox+ phages isolated from other strains of type B. tox- phages were also isolated, but they did not affect alpha toxin production. The tox+ phages also caused a marked change in the colonial morphology of type B strains. In this report we present evidence that alpha toxin production by C. novyi type A strain 5771 and type B strain 8024 depends upon the continued presence and participation of specific bacteriophages designated as NA1tox+ and NB1tox+, respectively.

  20. Use of botulinum toxin for voiding dysfunction

    Science.gov (United States)

    Dasgupta, Prokar

    2017-01-01

    The use of botulinum toxin A (BoNT-A) has expanded across a range of lower urinary tract conditions. This review provides an overview of the current indications for BoNT-A in the lower urinary tract and critically evaluates the published evidence within each area. The classic application of BoNT-A has been in the management of refractory neurogenic detrusor overactivity (NDO) and overactive bladder (OAB). There is a large volume of high-quality evidence, including numerous randomized placebo-controlled trials, which demonstrate the efficacy of BoNT-A over a long follow-up period. The culmination of this robust evidence-base has led to onabotulinumtoxin A (onaBoNT-A) receiving regulatory approval as a second-line treatment for NDO at a dose of 200 U and OAB at dose of 100 U. Other applications for BoNT-A are used on an off-license basis and include interstitial cystitis/bladder pain syndrome (IC/BPS), benign prostatic hyperplasia (BPH), and detrusor sphincter dyssynergia (DSD). These applications are associated with a less mature evidence-base although the literature is rapidly evolving. At present, the results for painful bladder syndrome (PBS) are promising and BoNT-A injections are recommended as a fourth line option in recent international guidelines, although larger randomized study with longer follow-up are required to confirm the initial findings. As a treatment for DSD, BoNT-A injections have shown potential but only in a small number of trials of limited quality. No definite recommendation can be made based on the current evidence. Finally, the results for the treatment of BPH have been variable and recent high quality randomized controlled trials (RCTs) have suggested no benefit over placebo so at present it cannot be recommended for routine clinical practice. Future advances of BoNT-A include liposome encapsulated formulations which are being developed as an alternative to intravesical injections. PMID:28540231

  1. A Medical Research and Evaluation Facility (MREF) and Studies Supporting the Medical Chemical Defense Program. Evaluation of the Passive Protection Against Five Serotypes of Botulinum Toxin Provided by Botulinum Human Immune Globulin in an Animal Model

    National Research Council Canada - National Science Library

    Olson, Carl

    1998-01-01

    Pentavalent (ABCDE) botulinum toxoid vaccine is intended for use as a prophylactic measure to protect combat troops against the lethal effects of botulinum toxins A-E, a group of toxins considered to be a serious biological warfare threat...

  2. Use of intradermal botulinum toxin to reduce sebum production and facial pore size.

    Science.gov (United States)

    Shah, Anil R

    2008-09-01

    Review the safety profile and subjective efficacy of intradermal botulinum toxin type A in facial pore size and sebum production. Retrospective analysis of 20 patients. Twenty consecutive patients with a single application of intradermal botulinum toxin type A were examined: Patients (17/20) noted an improvement in sebum production and a decrease in pores size at 1 month after injection. No complications were observed, and 17/20 patients were satisfied with the procedure. Preliminary data suggests that intradermal botulinum toxin may play a role in decreasing sebum production. Further quantitive study may be necessary to determine effects of intradermal botulinum toxin on pore size.

  3. Clostridium difficile and pediatric inflammatory bowel disease

    DEFF Research Database (Denmark)

    Martinelli, Massimo; Strisciuglio, Caterina; Veres, Gabor

    2014-01-01

    BACKGROUND: Clostridium difficile infection is associated with pediatric inflammatory bowel disease (IBD) in several ways. We sought to investigate C. difficile infection in pediatric patients with IBD in comparison with a group of children with celiac disease and to evaluate IBD disease course o...

  4. Isolation of Clostridium tetani from anaerobic empyema.

    Science.gov (United States)

    Mayall, B C; Snashall, E A; Peel, M M

    1998-11-01

    We report the isolation of Clostridium tetani (along with Fusobacterium mortiferum) from empyema pus. The patient, a 68 year old retired farmer from rural NSW, had recently undergone cholecystectomy, had heart failure and developed an empyema. He improved after drainage of the empyema and penicillin therapy, but died suddenly during convalescence.

  5. Clostridium difficile infection : epidemiology, complications and recurrences

    NARCIS (Netherlands)

    Bauer, Martijn Philippe

    2014-01-01

    Clostridium difficile is a spore-forming bacterium, the toxin-producing strains of which cause colitis. Risk factors are antibiotics, advanced age and severe comorbidity. C. difficile infection (CDI) has been regarded as mostly a hospital-acquired infection. Preventing relapses is considered the

  6. Genetic Diversity of Clostridium sporogenes PA 3679 Isolates Obtained from Different Sources as Resolved by Pulsed-Field Gel Electrophoresis and High-Throughput Sequencing.

    Science.gov (United States)

    Schill, Kristin M; Wang, Yun; Butler, Robert R; Pombert, Jean-François; Reddy, N Rukma; Skinner, Guy E; Larkin, John W

    2016-01-01

    Clostridium sporogenes PA 3679 is a nonpathogenic, nontoxic model organism for proteolytic Clostridium botulinum used in the validation of conventional thermal food processes due to its ability to produce highly heat-resistant endospores. Because of its public safety importance, the uncertain taxonomic classification and genetic diversity of PA 3679 are concerns. Therefore, isolates of C. sporogenes PA 3679 were obtained from various sources and characterized using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. The phylogenetic relatedness and genetic variability were assessed based on 16S rRNA gene sequencing and whole-genome single nucleotide polymorphism (SNP) analysis. All C. sporogenes PA 3679 isolates were categorized into two clades (clade I containing ATCC 7955 NCA3679 isolates 1961-2, 1990, and 2007 and clade II containing PA 3679 isolates NFL, UW, FDA, and Campbell and ATCC 7955 NCA3679 isolate 1961-4). The 16S maximum likelihood (ML) tree clustered both clades within proteolytic C. botulinum strains, with clade I forming a distinct cluster with other C. sporogenes non-PA 3679 strains. SNP analysis revealed that clade I isolates were more similar to the genomic reference PA 3679 (NCTC8594) genome (GenBank accession number AGAH00000000.1) than clade II isolates were. The genomic reference C. sporogenes PA 3679 (NCTC8594) genome and clade I C. sporogenes isolates were genetically distinct from those obtained from other sources (University of Wisconsin, National Food Laboratory, U.S. Food and Drug Administration, and Campbell's Soup Company). Thermal destruction studies revealed that clade I isolates were more sensitive to high temperature than clade II isolates were. Considering the widespread use of C. sporogenes PA 3679 and its genetic information in numerous studies, the accurate identification and genetic characterization of C. sporogenes PA 3679 are of critical importance. Copyright © 2015, American Society for Microbiology. All

  7. Botulinum Toxin for the Treatment of Tremor and Tics.

    Science.gov (United States)

    Lotia, Mitesh; Jankovic, Joseph

    2016-02-01

    The therapeutic applications of botulinum toxin (BoNT) have grown manifold since its initial approval in 1989 by the U.S. Food and Drug Administration for the treatment of strabismus, blepharospasm, and other facial spasms. Although it is the most potent biologic toxin known to man, long-term studies have established its safety in the treatment of a variety of neurologic and nonneurologic disorders. Despite a paucity of randomized controlled trials, BoNT has been found to be beneficial in treating a variety of tremors and tics when used by clinicians skilled in the administration of the drug for these hyperkinetic movement disorders. Botulinum toxin injections can provide meaningful improvement in patients with localized tremors and tics; in some cases, they may be an alternative to other treatments with more undesirable adverse effects. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  8. Interaction of Botulinum Toxin with the Epithelial Barrier

    Directory of Open Access Journals (Sweden)

    Yukako Fujinaga

    2010-01-01

    Full Text Available Botulinum neurotoxin (BoNT is a protein toxin (~150 kDa, which possesses a metalloprotease activity. Food-borne botulism is manifested when BoNT is absorbed from the digestive tract to the blood stream and enters the peripheral nerves, where the toxin cleaves core proteins of the neuroexocytosis apparatus and elicits the inhibition of neurotransmitter release. The initial obstacle to orally ingested BoNT entering the body is the epithelial barrier of the digestive tract. Recent cell biology and molecular biology studies are beginning to elucidate the mechanism by which this large protein toxin crosses the epithelial barrier. In this review, we provide an overview of the structural features of botulinum toxins (BoNT and BoNT complex and the interaction of these toxins with the epithelial barrier.

  9. Treatment of chronic tension-type headache with botulinum toxin: a double-blind, placebo-controlled clinical trial

    NARCIS (Netherlands)

    Padberg, M.; de Bruijn, S. F. T. M.; de Haan, R. J.; Tavy, D. L. J.

    2004-01-01

    Botulinum toxin is increasingly advocated as effective treatment in chronic tension-type headache. We conducted a randomized, placebo-controlled clinical trial to prove efficacy of botulinum toxin in chronic tension-type headache. Patients were randomly assigned to receive botulinum toxin (maximum

  10. Botulinum toxin: An emerging therapy in female bladder outlet obstruction

    Directory of Open Access Journals (Sweden)

    Aditya A Pradhan

    2009-01-01

    Discussion: There is a gradual improvement in symptoms over time and the maximal effect occurred at 10-14 days. The duration of improvement was approximately 16.8 weeks. All patients were satisfied by the degree of improvement felt. Conclusions: Botulinum toxin proved successful in improving the voiding characteristics. It possibly acts at the zone of hypertonicity at the bladder neck or midurethra. The only disadvantage is the high cost of the drug.

  11. Evaluation of neutralizing antibodies to type A, B, E, and F botulinum toxins in sera from human recipients of botulinum pentavalent (ABCDE) toxoid.

    Science.gov (United States)

    Siegel, L S

    1989-08-01

    Twenty-five serum specimens from personnel immunized with botulinum pentavalent toxoid (ABCDE) had titers of neutralizing antibodies to type A (5.7 to 51.6 IU/ml), type B (0.75 to 18 IU/ml), and type E (0.61 to 10 IU/ml) botulinum toxins. Titers for one type could not be used to predict titers for another type in individuals receiving the toxoid. Cross-neutralizing antibodies to type F botulinum toxin were not detected (less than 0.0125 IU/ml).

  12. Botulinum toxin injection of spastic finger flexors in hemiplegic patients.

    Science.gov (United States)

    Rodriquez, A A; McGinn, M; Chappell, R

    2000-01-01

    To assess the outcomes of botulinum toxin injection of spastic finger flexors followed by intensive training of finger extensors. Fourteen subjects with chronic hemiplegia spasticity of the upper limb had electromyographic-guided botulinum toxin injection into the long finger flexors. All patients presented with minimal active finger extension with the wrist flexed, sustained clonus of the finger flexors, functional proximal arm function, and absence of fixed contracture. Cadaver dissections directed selection of two injection sites: the flexor digitorum sublimis and the flexor digitorum profundus. Fifty mouse units of botulinum toxin were injected into each muscle. After injection, the subjects were instructed in a home program of stretching the long finger flexors, upper limb weight bearing with a weight-bearing splint, and exercise to improve finger extension control. Compared with preinjection measures, assessment the first week after the initial injection showed significantly reduced tone, reduced clonus, and greater active finger extension with the wrist in the neutral position. Four months later, the Ashworth scale increased to preinjection levels in the six subjects with repeated injections but was again decreased postinjection. Active finger extension with the wrist in the neutral position and clonus showed a statistically nonsignificant trend toward cumulative improvement after the second injection. The greatest change in finger extension and spasticity reduction occurred after the first injection. Continued significant improvement in finger extension was not observed.

  13. Strategies to decrease injection site pain in botulinum toxin therapy.

    Science.gov (United States)

    Paracka, Lejla; Kollewe, Katja; Wegner, Florian; Dressler, Dirk

    2017-10-01

    Botulinum toxin is now used for numerous indications including dystonias, spasticity, cerebral palsy, hyperhidrosis, cosmetics and chronic migraine. It has to be injected into its target tissues thus causing injection site pain. We wanted to compare the efficacy of various analgesic interventions suggested for reduction of injection site pain. In 13 healthy controls, pain thresholds in the fingertips II and III bilaterally were determined by the Mechanical Pain Threshold Test and the Repetitive Pain Stimulation Test at baseline and under nitrous oxide/oxygen, ice spray, local anaesthetic cream and forearm ischaemia. All interventions studied produce statistically significant and robust elevations of the pain threshold in both tests. Nitrous oxide/oxygen had stronger effects than the other interventions, although this superiority was statistically significant only in the Repetitive Pain Stimulation Test and not against ice spray. Also considering duration, localisation and penetration depth of analgesic effects, hyperhidrosis treatment may benefit from nitrous oxide/oxygen, ice spray and local anaesthetic cream. In palmar hyperhidrosis, forearm ischaemia is possible and also reduces botulinum toxin washout. Cosmetic indications may also benefit from nitrous oxide/oxygen and local anaesthetic cream. For botulinum toxin therapy of spasticity, dystonia and tremor, only nitrous oxide/oxygen may offer intramuscular analgesic effect. Its systemic and prolonged effect is also an advantage in injections in several body parts. Future studies are necessary to test the influence of penetration depth and combinations of analgesic interventions.

  14. Botulinum Toxin Treatment of Autonomic Disorders: Focal Hyperhidrosis and Sialorrhea.

    Science.gov (United States)

    Hosp, Christine; Naumann, Markus K; Hamm, Henning

    2016-02-01

    Primary focal hyperhidrosis is a common autonomic disorder that significantly impacts quality of life. It is characterized by excessive sweating confined to circumscribed areas, such as the axillae, palms, soles, and face. Less frequent types of focal hyperhidrosis secondary to underlying causes include gustatory sweating in Frey's syndrome and compensatory sweating in Ross' syndrome and after sympathectomy. Approval of onabotulinumtoxinA for severe primary axillary hyperhidrosis in 2004 has revolutionized the treatment of this indication. Meanwhile further type A botulinum neurotoxins like abobotulinumtoxinA and incobotulinumtoxinA, as well as the type B botulinum neurotoxin rimabotulinumtoxinB are successfully used off-label for axillary and various other types of focal hyperhidrosis. For unexplained reasons, the duration of effect differs considerably at different sites. Beside hyperhidrosis, botulinum neurotoxin is also highly valued for the treatment of sialorrhea affecting patients with Parkinson's disease, cerebral palsy, amyotrophic lateral sclerosis, motor neuron disease, and other neurologic conditions. With correct dosing and application, side effects are manageable and transient. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  15. Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E

    Directory of Open Access Journals (Sweden)

    Uma Basavanna

    2013-01-01

    Full Text Available The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA. The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it.

  16. DESTRUCTION OF FRANCISELLA TULARENSIS AND YERSINIA PESTIS PERSISTENCE OF BACILLUS ANTHRACIS SPORES AND CLOSTRIDIUM BOTULINUM IN MUNICIPAL SOLID LANDFILL LEACHATES

    Science.gov (United States)

    The United States Environmental Protection Agency Office of Research and Development National Homeland Security Research Center (NHSRC) in collaboration with the Department of Defense Edgewood Chemical Biological Center (ECBC) are evaluating the permanence of biological and chemi...

  17. Novel Treatments for Botulism: Development of Antagonists for Identified Steps in the Action of Botulinum Neurotoxins

    Science.gov (United States)

    1991-09-20

    J. (1989). ’Clues to the multi-phasic actions of botulinum toxins.’ XIV &me Confdrence en Neurobiologie , Gif-sur-Yvette, France. Dolly, J.0. (1990...mitter release by botulinum and tetanus toxins at Alyslia syn- apses: role(s) of the constituent chains’. XIV 6me Confdrence en Neurobiologie , Gif-sur

  18. Acute deterioration of bulbar function after botulinum toxin treatment for sialorrhoea in amyotrophic lateral sclerosis.

    NARCIS (Netherlands)

    Meijer, J.W.; Kuijk, A.A. van; Geurts, A.C.H.; Schelhaas, H.J.; Zwarts, M.J.

    2008-01-01

    Transcutaneous botulinum toxin injection in the salivary glands was introduced in 2000 as a new treatment for sialorrhoea in amyotrophic lateral sclerosis (ALS). We describe an ALS patient who developed serious complications of botulinum toxin treatment for sialorrhoea, and we review the relevant

  19. Review article: botulinum toxin in the therapy of gastrointestinal motility disorders.

    Science.gov (United States)

    Gui, D; Rossi, S; Runfola, M; Magalini, S C

    2003-07-01

    Since 1980, botulinum toxin has been employed for the treatment of various voluntary muscle spastic disorders in the fields of neurology and ophthalmology. More recently, botulinum toxin has been proved to be effective in the therapy of dyskinetic smooth muscle disorders of the gastrointestinal tract. Achalasia and anal fissure are the gastrointestinal disorders in which botulinum toxin therapy has been most extensively investigated. Botulinum toxin is the best treatment option for achalasia in patients whose condition makes them unfit for pneumatic dilation or surgery. In anal fissure, botulinum toxin is highly effective and may become the treatment of choice. In the future, botulinum toxin application in the gastrointestinal tract will be extended to many other gastrointestinal disorders, such as non-achalasic motor disorders of the oesophagus, dysfunction of Oddi's sphincter, achalasia of the internal anal sphincter and others. This article describes the mechanism of action, rationale of employment, indications and side-effects of botulinum toxin application in smooth muscle disorders of the gastrointestinal tract, and compares the results of different techniques of botulinum toxin therapeutic application.

  20. Identification of novel linear megaplasmids carrying a ß-lactamase gene in neurotoxigenic Clostridium butyricum type E strains.

    Directory of Open Access Journals (Sweden)

    Giovanna Franciosa

    Full Text Available Since the first isolation of type E botulinum toxin-producing Clostridium butyricum from two infant botulism cases in Italy in 1984, this peculiar microorganism has been implicated in different forms of botulism worldwide. By applying particular pulsed-field gel electrophoresis run conditions, we were able to show for the first time that ten neurotoxigenic C. butyricum type E strains originated from Italy and China have linear megaplasmids in their genomes. At least four different megaplasmid sizes were identified among the ten neurotoxigenic C. butyricum type E strains. Each isolate displayed a single sized megaplasmid that was shown to possess a linear structure by ATP-dependent exonuclease digestion. Some of the neurotoxigenic C. butyricum type E strains possessed additional smaller circular plasmids. In order to investigate the genetic content of the newly identified megaplasmids, selected gene probes were designed and used in Southern hybridization experiments. Our results revealed that the type E botulinum neurotoxin gene was chromosome-located in all neurotoxigenic C. butyricum type E strains. Similar results were obtained with the 16S rRNA, the tetracycline tet(P and the lincomycin resistance protein lmrB gene probes. A specific mobA gene probe only hybridized to the smaller plasmids of the Italian C. butyricum type E strains. Of note, a ß-lactamase gene probe hybridized to the megaplasmids of eight neurotoxigenic C. butyricum type E strains, of which seven from clinical sources and the remaining one from a food implicated in foodborne botulism, whereas this ß-lactam antibiotic resistance gene was absent form the megaplasmids of the two soil strains examined. The widespread occurrence among C. butyricum type E strains associated to human disease of linear megaplasmids harboring an antibiotic resistance gene strongly suggests that the megaplasmids could have played an important role in the emergence of C. butyricum type E as a human

  1. Septic arthritis due to Clostridium ramosum.

    Science.gov (United States)

    García-Jiménez, Antonio; Prim, Núria; Crusi, Xavier; Benito, Natividad

    2016-04-01

    Clostridium species are anaerobic bacilli that are rarely reported as etiologic agents of infectious arthritis. Previous cases of arthritis caused by Clostridium ramosum have not been reported. We describe the first 2 cases of C. ramosum arthritis. We reviewed the etiology of arthritis in our hospital during the previous 15 years. Both patients had underlying immunocompromising conditions and their infections involved a joint with preexisting disease: patient 1 had rheumatic arthritis and a prosthetic joint; patient 2, chronic renal failure on dialysis and hip osteoarthritis. The infection was hematogenously acquired and the course was indolent but destructive in both the cases. Management included open arthrotomy and resection arthroplasty. The infection had a persisting and relapsing course, and prolonged antibiotic treatment was required. In the literature review, we found 55 previous cases of arthritis caused by Clostridium species between 1966 and 2014; Clostridium perfringens was the most common infecting species; the infection was traumatically acquired in most of the cases. A total of 15 patients have been described with infections caused by C. ramosum; none had septic arthritis. The majority were elderly or immunocompromised adults. Proper collection, transportation and processing of clinical specimens is essential for diagnosing clostridial infections. More information about the best management of clostridial arthritis are needed. We describe the first 2 cases of septic arthritis caused by C. ramosum. They shared several pathogenic and clinical features. The possibility of anaerobic arthritis should always be considered when collecting diagnostic specimens. An increasing number of clostridial arthritis cases are likely to be diagnosed in future years. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Parameters Affecting Solvent Production by Clostridium pasteurianum

    OpenAIRE

    Dabrock, Birgit; Bahl, Hubert; Gottschalk, Gerhard

    1992-01-01

    The effect of pH, growth rate, phosphate and iron limitation, carbon monoxide, and carbon source on product formation by Clostridium pasteurianum was determined. Under phosphate limitation, glucose was fermented almost exclusively to acetate and butyrate independently of the pH and growth rate. Iron limitation caused lactate production (38 mol/100 mol) from glucose in batch and continuous culture. At 15% (vol/vol) carbon monoxide in the atmosphere, glucose was fermented to ethanol (24 mol/100...

  3. Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Nuemket, Nipawan [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Yoshikazu [Creative Research Institution ' Sousei,' Hokkaido University, Sapporo 001-0021 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tsukamoto, Kentaro; Tsuji, Takao [Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192 (Japan); Nakamura, Keiji; Kozaki, Shunji [Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531 (Japan); Yao, Min [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Isao, E-mail: tanaka@castor.sci.hokudai.ac.jp [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan)

    2011-07-29

    Highlights: {yields} We determined the crystal structure of the receptor binding domain of BoNT in complex with 3'-sialyllactose. {yields} An electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. {yields} Alanine site-directed mutagenesis showed that GBS and GBL are important for ganglioside binding. {yields} A cell binding mechanism, which involves cooperative contribution of two sites, was proposed. -- Abstract: Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 A. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.

  4. Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism

    International Nuclear Information System (INIS)

    Nuemket, Nipawan; Tanaka, Yoshikazu; Tsukamoto, Kentaro; Tsuji, Takao; Nakamura, Keiji; Kozaki, Shunji; Yao, Min; Tanaka, Isao

    2011-01-01

    Highlights: → We determined the crystal structure of the receptor binding domain of BoNT in complex with 3'-sialyllactose. → An electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. → Alanine site-directed mutagenesis showed that GBS and GBL are important for ganglioside binding. → A cell binding mechanism, which involves cooperative contribution of two sites, was proposed. -- Abstract: Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 A. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.

  5. PCR multiplex para identificação de isolados de Clostridium chauvoei e Clostridium septicum

    OpenAIRE

    Assis,R.A.; Lobato,F.C.F.; Lobato,Z.I.P.; Camargos,M.F.; Nascimento,R.A.P.; Vargas,A.P.C.; Salvarani,F.M.; Uzal,F.A.

    2008-01-01

    Padronizou-se uma técnica de reação em cadeia da polimerase múltipla (PCR multiplex) para detecção de Clostridium chauvoei e Clostridium septicum em culturas puras. Foram utilizados pares de iniciadores para segmentos específicos dos genes que codificam a flagelina de C. chauvoei e a toxina alfa de C. septicum. Para avaliaçã o da PCR multiplex, foram testados 16 isolados clínicos de C. chauvoei e 15 isolados de C. septicum provenientes de ruminantes, quatro sementes vacinais de cada um desses...

  6. New techniques for growing anaerobic bacteria: experiments with Clostridium butyricum and Clostridium acetobutylicum

    International Nuclear Information System (INIS)

    Adler, H.I.; Crow, W.D.; Hadden, C.T.; Hall, J.; Machanoff, R.

    1983-01-01

    Stable membrane fragments derived from Escherichia coli produce and maintain strict anaerobic conditions when added to liquid or solid bacteriological media. Techniques for growing Clostridium butyricum and Clostridium acetobutylicum in membrane-containing media are described. Liquid cultures initiated by very small inocula can be grown in direct contact with air. In solid media, colonies develop rapidly from individual cells even without incubation in anaerobic jars or similar devices. Observations on growth rates, spontaneous mutations, radiation, and oxygen sensitivity of anaerobic bacteria have been made using these new techniques

  7. Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Mingrui; Du, Yinming; Jiang, Wenyan; Chang, Wei-Lun; Yang, Shang-Tian [Ohio State Univ., Columbus, OH (United States). William G. Lowrie Dept. of Chemical and Biomolecular Engineering; Tang, I-Ching [Bioprocessing Innovative Company, Dublin, OH (United States)

    2012-01-15

    Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at {proportional_to}6.0. (orig.)

  8. Detection of botulinum neurotoxin serotype B at sub mouse LD(50 levels by a sandwich immunoassay and its application to toxin detection in milk.

    Directory of Open Access Journals (Sweden)

    Miles C Scotcher

    2010-06-01

    Full Text Available Botulinum neurotoxin (BoNT, the causative agent of botulism, a serious neuroparylatic disease, is produced by the anaerobic bacterium Clostridium botulinum and consists of a family of seven serotypes (A-H. We previously reported production of high-affinity monoclonal antibodies to BoNT serotype A.Recombinant peptide fragments of the light chain, the transmembrane and receptor-binding domains of the heavy chain of botulinum neurotoxin type B (BoNT/B were expressed in Escherichia coli as GST-fusion proteins and purified. These proteins were used to immunize BALB/cJ mice for the generation of monoclonal antibodies (mAbs. Antibody-producing hybridomas were detected using either a direct binding ELISA binding to plate-immobilized BoNT/B, or with a capture-capture ELISA whereby the capacity of the antibody to capture BoNT/B from solution was tested. A total of five mAbs were selected, two of which bound the toxin light chain and three bound the receptor-binding domain of BoNT/B heavy chain. MAb MCS6-27 was identified via capture-capture ELISA and was the only mAb able to bind BoNT/B in solution under physiological conditions. MAbs F24-1, F26-16, F27-33 and F29-40 were identified via direct binding ELISA, and were able to capture BoNT/B in solution only in the presence of 0.5-0.9 mM sodium dodecyl sulphate (SDS. MAb MCS6-27 and an anti-BoNT/B polyclonal antibody were incorporated into a sandwich ELISA that did not require SDS.We report here the generation of monoclonal antibodies to serotype B and the subsequent development of a sensitive sandwich immunoassay. This immunoassay has a detection limit of 100 fg BoNT/B, fifty times more sensitive than the mouse bioassay detection limit of 5 pg BoNT/B. Additionally, this assay detected as little as 39 pg/mL of toxin in skim, 2% and whole milk.

  9. Clostridium difficile: A healthcare-associated infection of unknown ...

    African Journals Online (AJOL)

    Clostridium difficile: A healthcare-associated infection of unknown significance in adults in sub-Saharan Africa. ... Abstract. Background: Clostridium difficile infection (CDI) causes a high burden of disease in high-resource healthcare systems, with significant morbidity, mortality, and financial implications. CDI is a ...

  10. Postpartum Clostridium sordellii infection associated with fatal toxic shock syndrome

    DEFF Research Database (Denmark)

    Rørbye, C; Petersen, Ina Sleimann; Nilas, Lisbeth

    2000-01-01

    Clostridium bacteria are anaerobic Gram positive spore-form-ing bacilli, known to cause distinct clinical syndromes such as botulism, tetanus, pseudomembranous colitis and myonecrosis. The natural habitats of Clostridium species are soil, water and the gastrointestinal tract of animals and humans....... sorlellii associated toxic shock syndrome - the first recognized in Scandinavia....

  11. Management of Clostridium difficile diarrhoea in District General ...

    African Journals Online (AJOL)

    Clostridium difficileis one of the most important causes of diarrhoea especially following antibiotic course. Elderly population is more susceptible and results in significant mortality and morbidity. We audited twenty four cases of Clostridium difficile in our hospital over duration of three months. We looked into the demographic

  12. Postpartum Clostridium sordellii infection associated with fatal toxic shock syndrome

    DEFF Research Database (Denmark)

    Rørbye, C; Petersen, Ina Sleimann; Nilas, Lisbeth

    2000-01-01

    Clostridium bacteria are anaerobic Gram positive spore-form-ing bacilli, known to cause distinct clinical syndromes such as botulism, tetanus, pseudomembranous colitis and myonecrosis. The natural habitats of Clostridium species are soil, water and the gastrointestinal tract of animals and humans....... Clostridium sordellii is rarely encountered in clinical specimens (1% of Clostridium species), but it has been described as a human pathogen with fatal potential. Two toxins, a lethal and a hemorrhagic (that antigenically and pathophysiologically appear similar to Clostridium difficile toxins B and A......, respectively) are responsible for this potential. Reviewing the obstetric literature, only six cases of postpartum endometritis caused by C. sordellii, are described - all being fatal. In addition, one lethal case of spontaneous endometritis resulting from C. sordellii is reported. The clinical aspects...

  13. Experience with botulinum toxin type A in medically intractable pediatric chronic daily headache.

    Science.gov (United States)

    Ahmed, Karman; Oas, Kimberly Hall; Mack, Kenneth J; Garza, Ivan

    2010-11-01

    In adults, botulinum toxin type A has been studied as a potentially effective treatment for chronic daily headache. For pediatric chronic daily headache, the literature evaluating efficacy of botulinum toxin type A is sparse, with no studies assessing tolerability. The purpose of this retrospective case series study was to assess tolerability and efficacy of botulinum toxin type A in the treatment of pediatric chronic daily headache. The series comprises 10 patients (ages 11-17 years) who received a standard 100-unit dose of onabotulinumtoxinA (trade name, Botox) for refractory chronic daily headache. Attention was given to therapeutic history, efficacy, and tolerability. The patients had attempted an average of 8.0 ± 2.40 S.D. therapies prior to botulinum toxin type A. Most patients reported adverse events from at least one of these prior medications. With botulinum toxin type A, four patients (40%) reported subjective but clinically meaningful relief, consisting of a decrease in headache intensity, and two patients additionally noted a decrease in headache frequency. The four responders noted improvements in quality of life. Three patients experienced minor adverse events from botulinum toxin type A. This case series suggests that botulinum toxin type A can be well tolerated and may be a useful therapeutic in pediatric patients with highly medically intractable chronic daily headache. Copyright © 2010 Elsevier Inc. All rights reserved.

  14. Botulinum neurotoxin type A in the masseter muscle: effects on incisor eruption in rabbits.

    Science.gov (United States)

    Navarrete, Alfonso L; Rafferty, Katherine L; Liu, Zi Jun; Ye, Wenmin; Greenlee, Geoffrey M; Herring, Susan W

    2013-04-01

    Botulinum neurotoxins are responsible for the paralytic food poisoning, botulism. Commercial formulations such as botulinum neurotoxin type A are increasingly used for various conditions, including cosmetic recontouring of the lower face by injection of the large masseter muscles. The paralysis of a major muscle of mastication lowers occlusal force and thus might affect tooth eruption. The purpose of this study was to investigate the effects of unilateral masseter muscle injection of botulinum neurotoxin type A on the rate of eruption of incisors in a rabbit model. We hypothesized that the teeth would overerupt in an underloaded environment. Forty rabbits were injected with either botulinum neurotoxin type A or saline solution in 1 masseter muscle. Mastication and muscle force production were monitored, and incisor eruption rate was assessed by caliper measurement of grooved teeth. The injection of saline solution had no effect. The masseter muscle injected with botulinum neurotoxin type A showed a dramatic loss of force 3 weeks after injection despite apparently normal mastication. Incisor eruption rate was significantly decreased for the botulinum neurotoxin type A group, an effect attributed to decreased attrition. This study has implications for orthodontics. Although findings from ever-growing rabbit incisors cannot be extrapolated to human teeth, it is clear that botulinum neurotoxin type A caused a decrease in bite force that could influence dental eruption. Copyright © 2013 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

  15. An overview of botulinum toxins: past, present, and future.

    Science.gov (United States)

    Cartee, Todd V; Monheit, Gary D

    2011-07-01

    Although the mechanism of action of botulinum toxin (BTX) has been intensively studied, many unanswered questions remain regarding the composition and clinical properties of the two formulations of BTX currently approved for cosmetic use. In the first half of this review, these questions are explored in detail, with emphasis on the most pertinent and revelatory studies in the literature. The second half delineates most of the common and some not so common uses of BTX in the face and neck, stressing important patient selection and safety considerations. Complications from neurotoxins at cosmetic doses are generally rare and usually technique dependent. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Advances in Assays and Analytical Approaches for Botulinum Toxin Detection

    Energy Technology Data Exchange (ETDEWEB)

    Grate, Jay W.; Ozanich, Richard M.; Warner, Marvin G.; Bruckner-Lea, Cindy J.; Marks, James D.

    2010-08-04

    Methods to detect botulinum toxin, the most poisonous substance known, are reviewed. Current assays are being developed with two main objectives in mind: 1) to obtain sufficiently low detection limits to replace the mouse bioassay with an in vitro assay, and 2) to develop rapid assays for screening purposes that are as sensitive as possible while requiring an hour or less to process the sample an obtain the result. This review emphasizes the diverse analytical approaches and devices that have been developed over the last decade, while also briefly reviewing representative older immunoassays to provide background and context.

  17. In Silico Analysis for the Study of Botulinum Toxin Structure

    Science.gov (United States)

    Suzuki, Tomonori; Miyazaki, Satoru

    2010-01-01

    Protein-protein interactions play many important roles in biological function. Knowledge of protein-protein complex structure is required for understanding the function. The determination of protein-protein complex structure by experimental studies remains difficult, therefore computational prediction of protein structures by structure modeling and docking studies is valuable method. In addition, MD simulation is also one of the most popular methods for protein structure modeling and characteristics. Here, we attempt to predict protein-protein complex structure and property using some of bioinformatic methods, and we focus botulinum toxin complex as target structure.

  18. A rapid qualitative assay for detection of Clostridium perfringens in canned food products.

    Science.gov (United States)

    Dave, Gayatri Ashwinkumar

    2017-01-01

    Clostridium perfringens (MTCC 1349) is a Gram-positive, anaerobic, endospore forming, and rod-shaped bacterium. This bacterium produces a variety of toxins under strict anaerobic environment. C. perfringens can grow at temperatures ranging between 20°C and 50°C. It is the major causetive agent for gas gangrene, cellulitis, septicemia, necrotic enteritis and food poisoning, which are common toxin induced conditions noted in human and animals. C. perfringens can produce produce four major types of toxins that are used for the classification of strains, classified under type A-E. Across the globe many countries, including the United States, are affected by C. perfringens food poisonings where it is ranked as one of the most common causes of food borne infections. To date, no direct one step assay for the detection of C. perfringens has been developed and only few methods are known for accurate detection of C. perfringens. Long detection and incubation time is the major consideration of these reporter assays. The prensent study proposes a rapid and reliable colorimetric assay for the detection of C. perfringens. In principale, this assay detects the para nitrophenyl (yellow colour end product) liberated due to the hydrolysis of paranitrophenyl phosphetidyl choline (PNPC) through phospholipase C (lecithinase). Constitutive secretion of phospholipase C is a charactristic feature of C. perfringens. This assay detects the presence of the extracellular lecithinse through the PNPC impragnated impregnated probe. The probe is impregnated with peranitrophenyl phosphotidyl choline ester, which is colourless substrate used by lecithinase. The designed assay is specific towards PNPC and detectes very small quantites of lecithinase under conditions used. The reaction is substrate specific, no cross reaction was observed upon incubation with other substrates. In addition, this assay gave negative results with other clostridium strains, no cross reactions were observed with other

  19. Botulinum toxin for spasticity in children with cerebral palsy: a comprehensive evaluation.

    Science.gov (United States)

    Bjornson, Kristie; Hays, Ross; Graubert, Cathy; Price, Robert; Won, Francine; McLaughlin, John F; Cohen, Morty

    2007-07-01

    Spasticity is a prevalent disabling clinical symptom for children with cerebral palsy. Treatment of spasticity with botulinum toxin in children with cerebral palsy was first reported in 1993. Botulinum toxin provides a focal, controlled muscle weakness with reduction in spasticity. Interpretation of the literature is difficult because of the paucity of reliable measures of spasticity and challenges with measuring meaningful functional changes in children with disabilities. This study documents the effects of botulinum toxin A injections into the gastrocnemius muscles in children with spastic diplegia. Outcomes are evaluated across all 5 domains of the National Centers for Medical and Rehabilitation Research domains of medical rehabilitation. A randomized, double-masked, placebo-controlled design was applied to 33 children with spastic diplegia with a mean age of 5.5 and Gross Motor Function Classification System Levels of I through III. Participants received either 12 U/kg botulinum toxin A or placebo saline injections to bilateral gastrocnemius muscles. Outcomes were measured at baseline and 3, 8, 12, and 24 weeks after injection. Significant decreases in the electromyographic representation of spasticity were documented 3 weeks after botulinum toxin A treatment. A significant decrease in viscoelastic aspects of spasticity was present at 8 weeks, and subsequent increases in dorsiflexion range were documented at 12 weeks for the botulinum toxin A group. Improvement was found in performance goals at 12 weeks and in maximum voluntary torque and gross motor function at 24 weeks for the botulinum toxin A. There were no significant differences between groups in satisfaction with performance goals, energy expenditure, Ashworth scores, or frequency of adverse effects. The safety profile of 12 U/kg of botulinum toxin A is excellent. Although physiologic and mechanical effects of treatment with botulinum toxin A were documented with functional improvement at 6 months

  20. Primary treatment of early fistula of parotid duct with botulinum toxin type A injection*

    Science.gov (United States)

    Ferron, Camila; Cernea, Selma Schuartz; de Almeida, Ada Regina Trindade; Cesar, Denise Vieira Galvão

    2017-01-01

    Salivary duct injury can be idiopathic, iatrogenic, or post-trauma and may result in sialocele or fistula. Most injuries regress spontaneously and botulinum toxin A is one of several therapeutic possibilities. We report a case of iatrogenic injury to the parotid duct after Mohs' micographic surgery for a squamous cell carcinoma excision in the left jaw region, treated by injection of botulinum toxin type A. Although the fistula by duct injury can be self-limiting, botulinum toxin injection by promoting the inactivity of the salivary gland allows rapid healing of the fistula. PMID:29364451

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

    Science.gov (United States)

    2011-10-01

    caused by ingestion of botulinum neurotoxin (BoNT) was first described as “ sausage poisoning” in 1820 and attributed to a bacterium in 1897 [1]. We...References. 1. Erbguth, F.J. and M. Naumann, Historical aspects of botulinum toxin: Justinus Kerner (1786-1862) and the " sausage poison...Liu, Analyzing a bioterror attack on the food supply: the case of botulinum toxin in milk. Proc Natl Acad Sci U S A, 2005. 102(28): p. 9984-9. 8

  2. The History of Collagenase Clostridium Histolyticum.

    Science.gov (United States)

    Yang, Kevin K; Bennett, Nelson

    2015-10-01

    After its U.S. FDA approval in 2013, Collagenase Clostridium histolyticum (CCh) has seen increasing use as a nonoperative treatment for Peyronie's disease (PD). We review the history of CCh and trials that led to its adoption. To provide a historical and contemporary context for the evolution of Collagenase Clostridium histolyticum as a treatment modality for Peyronie's disease. A comprehensive search of peer-reviewed literature was performed pertaining to CCh and its biochemical and clinical significance. The main outcome studied was the efficacy and safety profile of CCh in PD. CCh use in other diseases processes and its associated outcomes are also described. CCh injection yields objective improvement in penile curvature across multiple trials in PD patients. Recently, level 1 strength of evidence has emerged supporting its widespread use. As such, CCh stands as the only FDA-approved injectable therapy for PD. Adverse events were namely limited to local reactions. Serious systemic complications and need for intervention were rare. CCh is a safe and effective treatment for PD patients with deformities and plaque configuration amenable to injectable therapy. Multiple trials have demonstrated improvements in objective and subjective metrics such as penile curvature and bother scores. However, multiyear follow-up is needed to assess durability and its sustained clinical significance. Currently, refinement in dosing and technique has established a niche for CCh in PD patients who are affected by their symptoms but are not yet committed to surgical intervention. Yang KK and Bennett N. The history of collagenase clostridium histolyticum. Copyright © 2015 International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.

  3. Clostridium oedematiens: observations on potency assaying.

    Science.gov (United States)

    Macheak, M E

    1976-01-01

    The United States has established a Standard Requirement for the potency testing of biological products containing Clostridium oedematiens belonging to two types: type B (Cl. novyi) and type D (Cl. haemolyticum). Guinea pig testing has provided widely varying results depending on the origin of the animals. The tests reported are intended to determine the efficacity of the vaccines (Cl. novyi and Cl. haemolyticum) depending on the animal tested: guinea pigs, sheep and bovines; they further establish a parallelism between the antitoxin titer and the immunity of the animal.

  4. Regulation of Toxin Production in Clostridium perfringens

    Directory of Open Access Journals (Sweden)

    Kaori Ohtani

    2016-07-01

    Full Text Available The Gram-positive anaerobic bacterium Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tracts of humans and animals. C. perfringens causes gas gangrene and food poisoning, and it produces extracellular enzymes and toxins that are thought to act synergistically and contribute to its pathogenesis. A complicated regulatory network of toxin genes has been reported that includes a two-component system for regulatory RNA and cell-cell communication. It is necessary to clarify the global regulatory system of these genes in order to understand and treat the virulence of C. perfringens. We summarize the existing knowledge about the regulatory mechanisms here.

  5. Annotation of the Clostridium Acetobutylicum Genome

    Energy Technology Data Exchange (ETDEWEB)

    Daly, M. J.

    2004-06-09

    The genome sequence of the solvent producing bacterium Clostridium acetobutylicum ATCC824, has been determined by the shotgun approach. The genome consists of a 3.94 Mb chromosome and a 192 kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases, closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria.

  6. Clostridium difficile in Humans and Food Animals

    Centers for Disease Control (CDC) Podcasts

    2008-06-30

    Clostridium difficile is an antibiotic-resistant bacterium that causes diarrhea and sometimes serious intestinal illnesses. In recent years, C. difficile infections have been increasing in number and severity, including among some people outside healthcare settings. In this podcast, CDC's Dr. Michael Jhung discusses his recent study that looked at a new, increasingly prevalent strain of C. difficile in people and compared it to a strain historically found in animals to see whether the two might be linked. The study is published in the July 2008 issue of Emerging Infectious Diseases.  Created: 6/30/2008 by Emerging Infectious Diseases.   Date Released: 7/3/2008.

  7. Recent changes in Clostridium difficile infection

    Directory of Open Access Journals (Sweden)

    Moacyr Silva Júnior

    2012-03-01

    Full Text Available Clostridium difficile is the main cause of nosocomial diarrhea. Diarrhea associated with C. difficile has increased incidence, morbidity, and mortality in the last few years. The major related risk factors include use of antibiotics, elderly patients and prolonged hospital stay. Many patients receive combinations of antibiotics or multiple antibiotics, which represents the main risk to develop diarrhea associated to C. difficile or its recurrence. Therefore, interventions to improve antibiotic prescribing, as well as compliance with infection control measures can reduce hospital-acquired C. difficile infections. This review addresses the epidemiological changes in C. difficile disease and its treatment.

  8. Use of Botulinum toxin in 55 children with cerebral palsy

    Directory of Open Access Journals (Sweden)

    Mohammadi M

    2000-10-01

    Full Text Available Botulinum toxin A (BTA inhibits presynaptic release of acetylcholine at the neuromuscular junction and has reportedly been successful in the treatment of spastic disorders.To evaluate the effect of botulinum toxin on cerebral palsied children with spastic or mixed type of the disease, especially those patiens having spasticity as a cardinal symptom without joint contracture, we designed the following study. Ninety-one cases (55 of referred patients to pediatic Neurology outpatient clinics of children’s Medical Center were given BTA injections in affected muscles of the lower limb. They were reevaluating 3 to 5 weeks and 3 months later for type of walking and range of affected joints’ movement. The study showed a clinically significant gait improvement in 71.2% of patients (P<0.0005 and also an overall increased range of motion in affected limbs after BTA injection (P<0.04. Side effects occurred only in two cases as transient generalized weakness, gent recurvatum and ptosis. Drug effectiveness was time-limited, lasting abot 3 months in all patients ( a golden time for rehabilitation therapists to improve the patients’ condition. Overall, BTA has improved both the type of walking as well as the range of joints motion in our patients. So its’ administration is suggested in cerebral palsied children if the spasticity is a major and disabling sign

  9. Pneumatic dilation and botulinum toxin: when and why?

    Science.gov (United States)

    Bozzi, Rosamaria; Di Martino, Vincenzo; Inzirillo, Antonio; D'Avenia, Eugenio; Inzirillo, Maurizio; Cattaneo, Fabio; Cattaneo, Domenico

    2013-01-01

    Pneumatic dilation and botulinum toxin:when and why? The endoscopic treatment options of achalasia include botulinum toxin (BT) injection and pneumatic dilation (PD) of the lower esophageal sphincter (LES). BT can reduce the LES pressure by blocking the release of acetylcoline from presynaptic cholinergic nerve terminals in the myenteric plexus. Although the procedure is safe and good initial response is reported, there is a wide variability in the duration of the response and the effect tends to decrease over time. BT is usually recommended for elderly patients or patients with comorbid illnesses, who are poor candidates for more invasive procedures. PD aims at tearing the muscle fibers of the LES and is considered the most effective nonsurgical treatment for achalasia. Technical details of the procedure vary in different institutions and in many clinical settings the choice between PD or minimally invasive surgical myotomy depends upon local expertise in the procedures. Further endoscopic treatment options such as submucosal esophageal myotomy or self-expanding metallic stents are being studied.

  10. A Novel Inhibitor Prevents the Peripheral Neuroparalysis of Botulinum Neurotoxins

    Science.gov (United States)

    Azarnia Tehran, Domenico; Zanetti, Giulia; Leka, Oneda; Lista, Florigio; Fillo, Silvia; Binz, Thomas; Shone, Clifford C.; Rossetto, Ornella; Montecucco, Cesare; Paradisi, Cristina; Mattarei, Andrea; Pirazzini, Marco

    2015-01-01

    Botulinum neurotoxins (BoNTs) form a large class of potent and deadly neurotoxins. Given their growing number, it is of paramount importance to discover novel inhibitors targeting common steps of their intoxication process. Recently, EGA was shown to inhibit the action of bacterial toxins and viruses exhibiting a pH-dependent translocation step in mammalian cells, by interfering with their entry route. As BoNTs act in the cytosol of nerve terminals, the entry into an appropriate compartment wherefrom they translocate the catalytic moiety is essential for toxicity. Herein we propose an optimized procedure to synthesize EGA and we show that, in vitro, it prevents the neurotoxicity of different BoNT serotypes by interfering with their trafficking. Furthermore, in mice, EGA mitigates botulism symptoms induced by BoNT/A and significantly decreases the lethality of BoNT/B and BoNT/D. This opens the possibility of using EGA as a lead compound to develop novel inhibitors of botulinum neurotoxins. PMID:26670952

  11. Application of Purified Botulinum Type A Neurotoxin to Treat Experimental Trigeminal Neuropathy in Rats and Patients with Urinary Incontinence and Prostatic Hyperplasia

    Directory of Open Access Journals (Sweden)

    Yoshizo Matsuka

    2012-01-01

    Full Text Available Type A neurotoxin (NTX of Clostridium botulinum was purified by a simple procedure using a lactose gel column. The toxicity of this purified toxin preparation was retained for at least 1 year at −30°C by supplementation with either 0.1% albumin or 0.05% albumin plus 1% trehalose. When purified NTX was used to treat 49 patients with urinary incontinence caused by either refractory idiopathic or neurogenic detrusor overactivity, 36 patients showed significant improvement in symptoms. These beneficial effects were also observed in cases of prostatic hyperplasia. The results obtained with NTX were similar to that of Botox. The effects of NTX on trigeminal neuralgia induced by infraorbital nerve constriction (IoNC in rats were also studied. Trigeminal ganglion neurons from ipsilateral to IoNC exhibited significantly faster onset of FM4-64 release than sham-operated contralateral neurons. Intradermal injection of NTX in the area of IoNC alleviated IoNC-induced pain behavior and reduced the exaggerated FM4-64 release in trigeminal ganglion neurons.

  12. Secretion of clostridium cellulase by E. coli

    Science.gov (United States)

    Yu, Ida Kuo

    1998-01-01

    A gene, encoding an endocellulase from a newly isolated mesophilic Clostridium strain IY-2 which can digest bamboo fibers, cellulose, rice straw, and sawdust, was isolated by shotgun cloning in an E. coli expression plasmid pLC2833. E. coli positive clones were selected based on their ability to hydrolyze milled bamboo fibers and cellulose present in agar plates. One clone contained a 2.8 kb DNA fragment that was responsible for cellulase activity. Western blot analyses indicated that the positive clone produced a secreted cellulase with a mass of about 58,000 daltons that was identical in size to the subunit of one of the three major Clostridium cellulases. The products of cellulose digestion by this cloned cellulase were cellotetraose and soluble higher polymers. The cloned DNA contained signal sequences capable of directing the secretion of heterologous proteins from an E. coli host. The invention describes a bioprocess for the treatment of cellulosic plant materials to produce cellular growth substrates and fermentation end products suitable for production of liquid fuels, solvents, and acids.

  13. Key Research Issues in Clostridium difficile

    Directory of Open Access Journals (Sweden)

    George Zhanel

    2005-01-01

    Full Text Available Clostridium difficile is an emerging pathogen that causes C difficile-associated diarrhea, an important nosocomial infection. Control of this infection remains a challenge, and much needs to be determined about the antimicrobial resistance of the organism, antibiotic stewardship, contamination of the patient environment, and various host factors that determine susceptibility or resistance to infection. A national symposium focusing on C difficile infections, the Clostridium difficile Symposium on Emerging Issues and Research, was hosted on November 23, 2004, by the Department of Medical Microbiology and Infectious Diseases at the University of Manitoba, Winnipeg, Manitoba, in partnership with the Canadian Institutes of Health Research. This symposium, which aimed to summarize key research issues regarding C difficile infections in Canada, had the following objectives: to provide a forum for learning and discussion about C difficile and its impact on the health of Canadians; to identify the key research issues that should be addressed; and to explore potential research funding opportunities and collaboration. The present report summarizes key research issues identified for C difficile infections in Canada by addressing four major themes: diagnosis and surveillance, infection prevention and control, antibiotic stewardship, and clinical management.

  14. ETIOPATHOGENESIS OF DISEASES CAUSED BY CLOSTRIDIUM DIFFICILE

    Directory of Open Access Journals (Sweden)

    Predrag Stojanović

    2015-03-01

    Full Text Available Clostridium (C. difficile is a typical representative of the genus Clostridium. After colonization of the intestinal tract, toxigenic C. difficile strains are capable to produce two exotoxins, enterotoxin (toxin A and cytotoxin (toxin B, which cause diarrhea and colitis. Toxin A binds to specific carbohydrate receptors on the surface of intestinal cells and this is the beginning of damages in the intestinal tract which include destruction of the villi epithelium, limiting membrane, intercellular connections (zonula occludens and surface of the mucosa. If only toxin B is injected into intestinal cells, it does not cause damage nor increased fluids secretion. Probably, the reason for this is the inability of the toxin to bind to the cell membrane receptor in the intestinal tract under normal physiological conditions. Toxigenic strains of C. difficile can be found in the intestines of healthy people, without any symptoms or clinical signs (asymptomatic colonization. However, in people with risk factors, they can cause diarrhea of varying severity and life-threatening pseudomembranous colitis. These diseases are known as C. difficile associated disease - CDAD.

  15. Clostridium novyi, sordellii, and tetani: mechanisms of disease.

    Science.gov (United States)

    Aronoff, David M

    2013-12-01

    Clostridia represent a diverse group of spore-forming gram positive anaerobes that include several pathogenic species. In general, diseases caused by clostridia are a result of intoxication of the infected host. Thus, clostridial toxins have been targeted for diagnostic, therapeutic, and preventive strategies against infection. Studying the mechanisms of action of clostridial toxins has not only shed light on the pathogenesis of infection but has provided important new insights into cell biology and immunology. A primary purpose of this manuscript is to provide a succinct review on the mechanisms of disease caused by intoxication by the pathogens Clostridium tetani, Clostridium novyi, and Clostridium sordellii. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Botulinum toxin type A in the healing of chronic lesion following bilateral spasticity of gluteus muscle.

    Science.gov (United States)

    Cigna, Emanuele; Maruccia, Michele; Fanelli, Benedetta; Scuderi, Nicolò

    2014-08-01

    Use of botulinum toxin is expanding as the clinical studies demonstrate new potential therapeutic applications. In rehabilitation, botulinum toxin is above all used as adjunct therapy for the treatment of spasticity, but it may prove useful for other atypical clinical situations. A 17-year-old man had a sub-arachnoid haemorrhage following the rupture of cerebral aneurism. The patient presented gluteus maximus and medius bilaterally spasticity that produced a chronic lesion in the intergluteal cleft, a flexed wrist and a flexed elbow. As treatment for this spasticity, a total of 100 U botulinum toxin type A were injected into the glutei muscles. This treatment allowed for application of topical medication and subsequently, chronic lesion healing. Botulinum toxin A may be an important therapeutic aid for clinicians faced with treating persistent pathological conditions caused by spasticity. © 2012 The Authors. International Wound Journal © 2012 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  17. Combinatorial Strategies and High Throughput Screening in Drug Discovery Targeted to Channel of Botulinum

    National Research Council Canada - National Science Library

    Montel, Mauricio

    2004-01-01

    This program examines innovative approaches and powerful new technologies to identify selective and potent agents directed to prevent or relieve the neuroparalytic toxic actions of botulinum toxin A (BoNTA)1...

  18. Correction of glabellar protrusion after botulinum toxin injection for forehead wrinkles

    Directory of Open Access Journals (Sweden)

    Min Woo Kim

    2016-03-01

    Full Text Available Botulinum toxin blocks the release of the neurotransmitter acetylcholine and helps to treat wrinkles. The efficacy and safety of botulinum toxin injection for upper facial wrinkles were established in several previous studies. Common side effects include transient headache, bruising, eyebrow ptosis and eyelid swelling. No adverse life threatening events or long-term complications have been reported. However, there have been a few reports on site-specific side effects, except for eyebrow ptosis or eyelid swelling. Two previous cases showing exaggerated glabellar wrinkles after botulinum toxin injection for forehead horizontal lines reported that no specific treatment for the exaggeration of wrinkles were done and it was left to slowly recede. In the present report, we highlighted a patient, whose significant glabellar protrusion was rapidly and effectively corrected with the use of botulinum toxin injection.

  19. Dicty_cDB: Contig-U04536-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ulinum gene for infant neurotoxin type A. 44 5.1 1 ( X52066 ) Clostridium botulinum botA gene for type A neurotoxin.... 44 5.1 1 ( M30196 ) C.botulinum neurotoxin gene, complete cds. 44 5.1 1 ...( EU429475 ) Clostridium botulinum botulinum neurotoxin type A... 44 5.1 1 ( EU416227 ) Clostridium botulinu...inum strain A1/A2 boNT/A gene cl... 44 5.1 1 ( EF506573 ) Clostridium botulinum botulinum neurotoxin type A...... 44 5.1 1 ( EF506572 ) Clostridium botulinum botulinum neurotoxin type A... 44 5.1 1 ( EF470982 ) Clostrid

  20. In vitro inhibition of Clostridium difficile and Clostridium perfringens by commercial probiotic strains

    DEFF Research Database (Denmark)

    Schoster, A.; Kokotovic, Branko; Permin, Anders

    2013-01-01

    of this study was to examine the in vitro inhibitory effects of selected commercial bacterial strains on pathogenic clostridia and their growth characteristics under simulated gastrointestinal conditions.The inhibitory effects of 17 commercial strains of Lactobacillus (n = 16) and Bifidobacterium (n = 1......Probiotics have gained importance in human and veterinary medicine to prevent and control clostridial enteric disease. Limited information is available on the ability of different probiotic bacteria used in food products to inhibit Clostridium difficile and Clostridium perfringens. The objective......) on the reference strains of C. difficile and C. perfringens were assessed by an agar well diffusion assay and by a broth culture inhibition assay using cell-free supernatant harvested at different growth phases, with and without pH neutralization. To study growth characteristics, probiotic strains were cultivated...

  1. EPIDEMIOLOGIC INVESTIGATION OF CLOSTRIDIUM DIFFICILE AND CLOSTRIDIUM PERFRINGENS IN HEALTHY HORSES

    DEFF Research Database (Denmark)

    Schoster, Angelika; Arroyo, Luis; Staempfli, Henry

    Clostridium difficile and Clostridium perfringens are important causes of equine colitis but can also be found in healthy individuals. Epidemiologic information is restricted to cross-sectional studies of fecal shedding with little information on prevalence in gastrointestinal compartments other...... than feces and variability in shedding over time. The objectives were to investigate the presence of C. difficile and C. perfringens in healthy horses over time and assess prevalence in different gastrointestinal compartments. Feces were collected monthly from 25 horses for one year. Ingesta were...... collected from nine GI compartments of a separate group of 15 euthanized horses. Selective enrichment culture was performed, followed by toxin gene detection and ribotyping (C. difficile) and multiplex PCR (C. perfringens). Toxigenic C. difficile was isolated from 15/275 (5.5%) samples from 10/25 (40...

  2. Botulinum toxin type B blocks sudomotor function effectively: a 6 month follow up.

    Science.gov (United States)

    Birklein, Frank; Eisenbarth, Gabi; Erbguth, Frank; Winterholler, Martin

    2003-12-01

    This study analyzes the suppression of sweat gland activity by botulinum toxin type B. We injected botulinum toxin type B (between 2 and 1000 mouse units subcutaneously) in the lateral side of both lower legs in 15 healthy volunteers. Sweat tests were carried out before botulinum toxin type B injections, and at 3 wk, 3 mo, and 6 mo. We studied focal anhidrosis by iodine-starch staining and by capacitance hygrometry after carbachol iontophoresis, according to the quantitative sudomotor axon reflex test (QSART). Iodine starch staining indicated that a threshold dose of 8 mouse units botulinum toxin type B leads to anhidrotic skin spots (>4 cm2) after 3 wk. Duration of anhidrosis was prolonged for 3 mo when 15 mouse units and for 6 mo when 125 mouse units botulinum toxin type B were injected. The size of the anhidrotic area decreased with time (piodine-starch staining and QSART indicated that botulinum toxin type B suppresses sudomotor function effectively, in a concentration-dependent manner.

  3. Efficacy of botulinum toxins on bruxism: an evidence-based review.

    Science.gov (United States)

    Long, Hu; Liao, Zhengyu; Wang, Yan; Liao, Lina; Lai, Wenli

    2012-02-01

    The objective of this study was to assess the efficacy of botulinum toxins on bruxism. Electronic databases (PubMed, Embase and Science Citation Index), websites (Cochrane Central Register of Controlled Trials and ClinicalTrials.gov) and the literature database of SIGLE (System for Information on Grey Literature in Europe) were searched from January 1990 to April 2011 for randomised controlled trials or nonrandomised studies assessing the efficacy of botulinum toxins on bruxism. There was no language restriction. Through a predefined search strategy, we retrieved 28 studies from PubMed, 94 from Embase, 60 from the Science Citation Index, two ongoing clinical trials and two from the Cochrane Central Register of Controlled Trials. Of these, only four studies met our inclusion criteria and were finally included. Of the four included studies, two were randomised controlled trials and two were controlled before-and-after studies. These studies showed that botulinum toxin injections can reduce the frequency of bruxism events, decrease bruxism-induced pain levels and satisfy patients' self-assessment with regard to the effectiveness of botulinum toxins on bruxism. In comparison with oral splint, botulinum toxins are equally effective on bruxism. Furthermore, botulinum toxin injections at a dosage of bruxism and are safe to use. Therefore, they can be used clinically for otherwise healthy patients with bruxism. © 2012 FDI World Dental Federation.

  4. Effect of botulinum toxin A on proliferation and apoptosis in the T47D breast cancer cell line.

    Science.gov (United States)

    Bandala, Cindy; Perez-Santos, Jose Luis Martin; Lara-Padilla, Eleazar; Delgado Lopez, Guadalupe; Anaya-Ruiz, Maricruz

    2013-01-01

    The present study was performed to assess the activity of the botulinum toxin A on breast cancer cells. The T47D cell line was exposed to diverse concentrations of the botulinum toxin A and cell viability and apoptosis were estimated using MTT and propidium iodine/annexin V methods, respectively. Botulinum toxin A exerted greater cytotoxic activity in T47D cells in comparison with MCF10A normal cells; this appeared to be via apoptotic processes caspase-3 and -7. In conclusion, botulinum toxin A induces caspase-3 and -7 dependent apoptotic processes in the T47D breast cancer cell line.

  5. Toxina botulínica no tratamento da dor Toxina botulínica en el tratamiento del dolor Botulinum toxin in pain treatment

    Directory of Open Access Journals (Sweden)

    Orlando Carlos Gomes Colhado

    2009-06-01

    Full Text Available JUSTIFICATIVA E OBJETIVOS: A toxina botulínica (TxB, uma das mais potentes toxinas bacterianas conhecidas, tem reconhecidamente ação terapêutica eficaz no tratamento de algumas síndromes dolorosas. Entretanto, algumas de suas indicações ainda estão em fase de comprovação com relação a sua eficácia. O objetivo deste estudo foi revisar o histórico, propriedades farmacológicas e aplicações clinicas da TxB, quando empregada no tratamento de dores de diferentes origens. CONTEÚDO: A TxB é o produto da fermentação do Clostridium Botulinum, uma bactéria anaeróbia Gram-positiva. Comercialmente, as TxB existem nas formas A e B, agentes biológicos obtidos laboratorialmente. A TxB, uma neurotoxina que possui alta afinidade pelas sinapses colinérgicas, ocasiona bloqueio na liberação de acetilcolina pelo terminal nervoso, sem alterar a condução neural de sinais elétricos ou síntese e armazenamento de acetilcolina. Comprovadamente, a TxB pode enfraquecer seletivamente a musculatura dolorosa, interrompendo o ciclo espasmo-dor. Com relação à dor, varias publicações têm demonstrado a eficácia e segurança da TxB-A no tratamento da cefaleia tipo tensão, migrânea, dor lombar crônica e dor miofascial. CONCLUSÕES: A TxB-A é segura e bem tolerada em desordens dolorosas crônicas, onde regimes de farmacoterapia podem sabidamente provocar efeitos colaterais. Outra vantagem é a redução do uso de analgésicos e o tempo de ação de 3 a 4 meses por dose. Entretanto pesquisas futuras serão necessárias para se estabelecer a eficácia da TxB-A em desordens dolorosas crônicas e seu exato mecanismo no alivio da dor, bem como seu potencial em tratamentos multifatoriais.JUSTIFICATIVA Y OBJETIVOS: La toxina botulínica (TxB, una de las más potentes toxinas bacterianas de que se tiene conocimiento, posee una reconocida acción terapéutica eficaz en el tratamiento de algunos síndromes dolorosos. Sin embargo, algunas de sus

  6. Rectal bacteriotherapy for recurrent Clostridium difficile-associated diarrhoea

    DEFF Research Database (Denmark)

    Tvede, M; Tinggaard, M; Helms, M

    2015-01-01

    Clostridium difficile infection is one of the most common nosocomial infections. Among other alternatives to standard treatment with vancomycin for recurrent infection are faecal microbiota transplantation and rectal bacteriotherapy with a fixed mixture of intestinal bacterial strains isolated from...

  7. Clostridium Difficile-Associated Diarrhea in 200 Canadian Children

    Directory of Open Access Journals (Sweden)

    Véronique Morinville

    2005-01-01

    Full Text Available OBJECTIVE: Clostridium difficile-associated diarrhea is a major problem in adults. The present study was conducted to assess risk factors and outcomes in children with C difficile-associated diarrhea.

  8. First Report of Clostridium lavalense Isolated in Human Blood Cultures

    Directory of Open Access Journals (Sweden)

    Richard Garceau

    2016-01-01

    Full Text Available An 88-year-old man was admitted to the hospital with worsening malaise, fever, and weakness. Anaerobic blood culture bottles revealed the presence of an anaerobic, Gram-positive sporulated bacillus. Empirical antibiotherapy with intravenous piperacillin-tazobactam was initiated. The patient defervesced after four days and was switched to oral amoxicillin on his 6th day of antibiotic therapy and later discharged from the hospital. Four months later, he had recovered. The bacterium was initially identified as Clostridium butyricum using anaerobic manual identification panel. 16S rRNA gene sequence and phylogenetic analysis showed the bacterium to be Clostridium lavalense, a recently described species with no previously published case of isolation in human diagnostic samples so far. This is the first report of Clostridium lavalense isolation from human blood cultures. Further studies are needed in order to elucidate the role of Clostridium lavalense in human disease and its virulence factors.

  9. Comparison of media for enumeration of Clostridium perfringens from foods

    NARCIS (Netherlands)

    Jong, A.E.I. de; Eijhusen, G.P.; Brouwer-Post, E.J.F.; Grand, M.; Johansson, T.; Kärkkäinen, T.; Marugg, J.; Veld, P.H. in 't; Warmerdam, F.H.M.; Wörner, G.; Zicavo, A.; Rombouts, F.M.; Beumer, R.R.

    2003-01-01

    Many media have been developed to enumerate Clostridium perfringens from foods. In this study, six media [iron sulfite (IS) agar, tryptose sulfite cycloserine (TSC) agar, Shahidi Ferguson perfringens (SFP) agar, sulfite cycloserine azide (SCA), differential clostridial agar (DCA), and oleandomycin

  10. Clostridium difficile Infection Worsens the Prognosis of Ulcerative Colitis

    Directory of Open Access Journals (Sweden)

    María E Negrón

    2014-01-01

    Full Text Available BACKGROUND: The impact of Clostridium difficile infections among ulcerative colitis (UC patients is well characterized. However, there is little knowledge regarding the association between C difficile infections and postoperative complications among UC patients.

  11. Reactive arthritis induced by recurrent Clostridium difficile colitis

    Directory of Open Access Journals (Sweden)

    Allison Marr

    2012-01-01

    Full Text Available Clostridium difficile colitis is a common infection that can be difficult to resolve and may result in recurrent infections. Reactive arthritis is a rare presentation of this disease and its treatment is not well differentiated in the literature. We describe a case of reactive arthritis occurring in a patient with a history of recurrent Clostridium difficile colitis while currently receiving a taper of oral vancomycin. His arthritis symptoms resolved with corticosteroids and continued treatment with anticlostridial antibiotics.

  12. Clostridium difficile infection in Europe: a hospital-based survey

    DEFF Research Database (Denmark)

    Bauer, Martijn P; Notermans, Daan W; van Benthem, Birgit H B

    2011-01-01

    Little is known about the extent of Clostridium difficile infection in Europe. Our aim was to obtain a more complete overview of C difficile infection in Europe and build capacity for diagnosis and surveillance.......Little is known about the extent of Clostridium difficile infection in Europe. Our aim was to obtain a more complete overview of C difficile infection in Europe and build capacity for diagnosis and surveillance....

  13. [Botulinum toxin: An important complement for facial rejuvenation surgery].

    Science.gov (United States)

    Le Louarn, C

    2017-10-01

    The improved understanding of the functional anatomy of the face and of the action of the botulinum toxin A leads us to determine a new injection procedure which consequently decreases the risk of eyebrow and eyelid ptosis and increases the toxin's injection possibilities and efficiencies. With less units of toxin, the technique herein described proposes to be more efficient on more muscles: variable toxin injections concentration adapted to each injected muscle are used. Thanks to a new procedure in the upper face, toxin A injection can be quite close to an endoscopic surgical action. In addition, interesting results are achievable to rejuvenate the lateral canthus with injection on the upper lateral tarsus, to rejuvenate the nose with injection at the alar base, the jawline and the neck region. Lastly, a smoothing effect on the skin (meso botox) is obtained by the anticholinergic action of the toxin A on the dermal receptors. Copyright © 2017. Published by Elsevier Masson SAS.

  14. Botulinum Toxin Treatment of Spasticity in Adults and Children.

    Science.gov (United States)

    Moeini-Naghani, Iman; Hashemi-Zonouz, Taraneh; Jabbari, Bahman

    2016-02-01

    Spasticity is a frequent symptom in stroke, multiple sclerosis, cerebral or spinal trauma, and cerebral palsy that affects and disables a large number of adults and children. In this review, we discuss the pathophysiology and nonpharmacologic and pharmacologic treatments of spasticity with emphasis on the role of botulinum neurotoxins (BoNTs). The world literature is reviewed on double-blind and placebo-controlled clinical trials reporting safety and efficacy of BoNT treatment in adult spasticity and spasticity of children with cerebral palsy. The evidence for efficacy is presented from recommendations of the Assessment and Therapeutics subcommittee of the American Academy of Neurology. A technical section describes the techniques and recommended doses of BoNTs in spasticity. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  15. Botulinum toxin treatment for limb spasticity in childhood cerebral palsy

    Directory of Open Access Journals (Sweden)

    Vito ePavone

    2016-02-01

    Full Text Available CP is the most common cause of chronic disability in childhood occurring in 2 to 2.5/1000 births. It is a severe disorder and a significant number of patients present cognitive delay and difficulty in walking. The use of botulinum toxin (BTX has become a popular treatment for CP especially for spastic and dystonic muscles while avoiding deformity and pain. Moreover, the combination of physiotherapy, casting, orthotics and injection of BTX may delay or decrease the need for surgical intervention while reserving single-event, multi-level surgery for fixed musculotendinous contractures and bony deformities in older children. This report highlights the utility of BTX in the treatment of cerebral palsy in children. We include techniques for administration, side effects and possible resistance as well as specific use in the upper and lower limbs muscles

  16. Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

    Energy Technology Data Exchange (ETDEWEB)

    Schmitt, John; Karalewitz, Andrew; Benefield, Desire A.; Mushrush, Darren J.; Pruitt, Rory N.; Spiller, Benjamin W.; Barbieri, Joseph T.; Lacy, D. Borden (Vanderbilt); (MCW)

    2010-10-19

    Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of synaptotagmin I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only SytI. Both serotypes bind ganglioside G{sub T1b}. The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform for understanding the physiological relevance of different Syt receptor isoforms in vivo.

  17. Peptide inhibitors of botulinum neurotoxin by mRNA display

    International Nuclear Information System (INIS)

    Yiadom, Kwabena P.A.B.; Muhie, Seid; Yang, David C.H.

    2005-01-01

    Botulinum neurotoxins (BoNTs) are extremely toxic. The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion. Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs. Toward that goal, we produced a synthetic cDNA for the expression and purification of the metalloprotease of BoNT/A in Escherichia coli as a biotin-ubiquitin fusion protein, and constructed a combinatorial peptide library to screen for BoNT/A light chain inhibitors using mRNA display. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs

  18. Sixth nerve palsy following botulinum toxin injection for facial rejuvenation.

    Science.gov (United States)

    Dolar Bilge, Ayse; Sadigov, Fariz; Salar-Gomceli, Senem

    2017-06-01

    Botulinum toxin A (BTX) has been widely used for a variety of facial esthetic procedures within the last couple of decades. Efficacy and safety of BTX for facial rejuvenation has been extensively studied in multiple randomized prospective controlled trials. Focal weakness is among the most commonly reported adverse effects. Adverse reactions tend to occur most commonly due to errors in dosing formulation and errors with the techniques of the application. No serious long-term complications have been reported. We present the case of a 52-year-old female presenting with diplopia one week following the injection of BTX for facial rejuvenation at glabella, forehead and crow's feet areas. Injection of BTX adjacent to periorbital area may be associated with extra-ocular muscle paralysis.

  19. [New aspects on Clostridium difficile infection].

    Science.gov (United States)

    von Müller, Lutz

    2016-08-01

    Clostridium difficile infection (CDI) is a frequent and complex disease which is influenced by the repertoire of bacterial virulence factors, by host immunity and by the intestinal microbiome. These complex interaction opens a number of options which may be used for treatment in the future. One example for new treatment options is fecal microbiota transplantation (FMT). Driven by C. difficile related research activities the knowledge of protective microorganism is increasing and it may be assumed that bacteriotherapy by next-generation probiotics may be used very soon also for other diseases. Very often, CDI reflects to the clinician that antibiotic therapy is associated with side effects. Therefore, C. difficile is the guilty conscience which helps to implement targeted and restrictive antibiotic use in the daily practice. © Georg Thieme Verlag KG Stuttgart · New York.

  20. Investigational new treatments for Clostridium difficile infection.

    Science.gov (United States)

    Ivarsson, Mattias E; Leroux, Jean-Christophe; Castagner, Bastien

    2015-05-01

    Significant progress has been made by industry and academia in the past two years to address the medical threats posed by Clostridium difficile infection. These developments provide an excellent example of how patient need has driven a surge of innovation in drug discovery. Indeed, only two drugs were approved for the infection in the past 30 years but there are 13 treatment candidates in clinical trials today. What makes the latter number even more remarkable is the diversity in the strategies represented (antibiotics, microbiota supplements, vaccines, antibiotic quenchers and passive immunization). In this review, we provide a snapshot of the current stage of these breakthroughs and argue that there is still room for further innovation in treating C. difficile infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Engineering clostridium strain to accept unmethylated DNA.

    Directory of Open Access Journals (Sweden)

    Hongjun Dong

    Full Text Available It is difficult to genetically manipulate the medically and biotechnologically important genus Clostridium due to the existence of the restriction and modification (RM systems. We identified and engineered the RM system of a model clostridial species, C. acetobutylicum, with the aim to allow the host to accept the unmethylated DNA efficiently. A gene CAC1502 putatively encoding the type II restriction endonuclease Cac824I was identified from the genome of C. acetobutylicum DSM1731, and disrupted using the ClosTron system based on group II intron insertion. The resulting strain SMB009 lost the type II restriction endonuclease activity, and can be transformed with unmethylated DNA as efficiently as with methylated DNA. The strategy reported here makes it easy to genetically modify the clostridial species using unmethylated DNA, which will help to advance the understanding of the clostridial physiology from the molecular level.

  2. Biotechnological potential of Clostridium butyricum bacteria

    Directory of Open Access Journals (Sweden)

    Daria Szymanowska-Powałowska

    2014-09-01

    Full Text Available In response to demand from industry for microorganisms with auspicious biotechnological potential, a worldwide interest has developed in bacteria and fungi isolation. Microorganisms of interesting metabolic properties include non-pathogenic bacteria of the genus Clostridium, particularly C. acetobutylicum, C. butyricum and C. pasteurianum. A well-known property of C. butyricum is their ability to produce butyric acid, as well as effectively convert glycerol to 1,3-propanediol (38.2 g/L. A conversion rate of 0.66 mol 1,3-propanediol/mol of glycerol has been obtained. Results of the studies described in the present paper broaden our knowledge of characteristic features of C. butyricum specific isolates in terms of their phylogenetic affiliation, fermentation capacity and antibacterial properties.

  3. Biosynthesis of butyric acid by Clostridium tyrobutyricum.

    Science.gov (United States)

    Huang, Jin; Tang, Wan; Zhu, Shengquan; Du, Meini

    2018-03-21

    Butyric acid (C 3 H 7 COOH) is an important chemical that is widely used in foodstuffs along with in the chemical and pharmaceutical industries. The bioproduction of butyric acid via large-scale fermentation has the potential to be more economical and efficient than petrochemical synthesis. In this paper, the metabolic pathways involved in the production of butyric acid from Clostridium tyrobutyricum using hexose and pentose as substrates are investigated, and approaches to enhance butyric acid production via genetic modification are discussed. Finally, bioreactor modifications (including fibrous bed bioreactor, inner disc-shaped matrix bioreactor, fibrous matrix packed in porous levitated sphere carriers), low-cost feedstocks and special treatments (including continuous fermentation with cell recycling, extractive fermentation with solvent, using different artificial electron carriers) intended to improve the feasibility of commercial butyric acid bioproduction are summarized.

  4. Factors affecting autocatalysis of botulinum A neurotoxin light chain.

    Science.gov (United States)

    Ahmed, S Ashraf; Ludivico, Matthew L; Smith, Leonard A

    2004-10-01

    The light chain of botulinum neurotoxin serotype A undergoes autocatalytic fragmentation into two major peptides during purification and storage (Ahmed S. A. et al. 2001, J. Protein Chem. 20:221-231) by both intermolecular and intramolecular mechanisms (Ahmed S. A. et al. 2003, Biochemistry 42:12539 12549). In this study, we investigated the effects of buffers and salts on this autocatalytic reaction in the presence and absence of zinc chloride. In the presence of zinc chloride, the fragmentation reaction was enhanced in each of acetate, MES, HEPES and phosphate buffers with maximum occurring in acetate when compared to those in the absence of zinc chloride. Adding sodium chloride in phosphate buffer in the presence of zinc chloride increased the extent of proteolysis. Irrespective of the presence of zinc chloride, adding sodium chloride or potassium chloride in phosphate buffer elicited an additional proteolytic reaction. Higher concentrations of sodium phosphate buffer enhanced the autocatalytic reaction in the absence of zinc chloride. In contrast, in the presence of zinc chloride, higher concentrations of sodium phosphate decreased the autocatalytic reaction. Optimum pH of autocatalysis was not affected significantly by the absence or presence of zinc chloride. Like zinc chloride, other chlorides of divalent metals, such as magnesium, cobalt, iron and calcium also enhanced the autocatalytic reaction. Polyols such as ethylene glycol protected the light chain from fragmentation. Exposure of light chain to UV radiation led to enhanced fragmentation. In order to avoid fragmentation, the protein should be stored frozen in a low concentration buffer of neutral or higher pH devoid of any metal. Our results provide a choice of buffers and salts for isolation, purification and storage of intact botulinum neurotoxin serotype A light chain.

  5. Sphincter of Oddi botulinum toxin injection to prevent pancreatic fistula after distal pancreatectomy.

    Science.gov (United States)

    Hackert, Thilo; Klaiber, Ulla; Hinz, Ulf; Kehayova, Tzveta; Probst, Pascal; Knebel, Phillip; Diener, Markus K; Schneider, Lutz; Strobel, Oliver; Michalski, Christoph W; Ulrich, Alexis; Sauer, Peter; Büchler, Markus W

    2017-05-01

    Postoperative pancreatic fistula represents the most important complication after distal pancreatectomy. The aim of this study was to evaluate the use of a preoperative endoscopic injection of botulinum toxin into the sphincter of Oddi to prevent postoperative pancreatic fistula (German Clinical Trials Register number: DRKS00007885). This was an investigator-initiated, prospective clinical phase I/II trial with an exploratory study design. We included patients who underwent preoperative endoscopic sphincter botulinum toxin injection (100 units of Botox). End points were the feasibility, safety, and postoperative outcomes, including postoperative pancreatic fistula within 30 days after distal pancreatectomy. Botulinum toxin patients were compared with a control collective of patients undergoing distal pancreatectomy without botulinum toxin injection by case-control matching in a 1:1 ratio. Between February 2015 and February 2016, 29 patients were included. All patients underwent successful sphincter of Oddi botulinum toxin injection within a median of 6 (range 0-10) days before operation. One patient had an asymptomatic, self-limiting (48 hours) increase in serum amylase and lipase after injection. Distal pancreatectomy was performed in 24/29 patients; 5 patients were not resectable. Of the patients receiving botulinum toxin, 7 (29%) had increased amylase levels in drainage fluid on postoperative day 3 (the International Study Group of Pancreatic Surgery definition of postoperative pancreatic fistula grade A) without symptoms or need for reintervention. Importantly, no clinically relevant fistulas (International Study Group of Pancreatic Surgery grades B/C) were observed in botulinum toxin patients compared to 33% postoperative pancreatic fistula grade B/C in case-control patients (P pancreatectomy. The results of the present trial suggest its efficacy in the prevention of clinically relevant postoperative pancreatic fistula and are validated currently in the

  6. Characterisation of botulinum toxins type A and B, by matrix-assisted laser desorption ionisation and electrospray mass spectrometry

    NARCIS (Netherlands)

    Baar, B.L.M. van; Hulst, A.G.; Jong, A.L. de; Wils, E.R.J.

    2002-01-01

    A method earlier developed for the mass spectrometric (MS) identification of tetanus toxin (TTx) was applied to botulinum toxins type A and B (BTxA and BTxB). Botulinum toxins are extremely neurotoxic bacterial toxins, likely to be used as biological warfare agent. Biologically active BTxA and BTxB

  7. 76 FR 29752 - Nomination of In Vitro Test Methods for Detection and Quantification of Botulinum Neurotoxins and...

    Science.gov (United States)

    2011-05-23

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES Nomination of In Vitro Test Methods for Detection and Quantification of Botulinum Neurotoxins and Detection of Non-Endotoxin Pyrogens; Data Request for Substances... detecting and quantifying botulinum neurotoxin (BoNT), and (2) an in vitro test method proposed for...

  8. Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens.

    OpenAIRE

    Kim, A Y; Blaschek, H P

    1989-01-01

    A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Follo...

  9. Unilateral transient mydriasis and ptosis after botulinum toxin injection for a cosmetic procedure

    Directory of Open Access Journals (Sweden)

    Akkaya S

    2015-02-01

    Full Text Available Sezen Akkaya,1 Hatice Kübra Kökcen,1 Tuğba Atakan2 1Fatih Sultan Mehmet Education and Training Hospital, Ophthalmology Clinics, Bostanci, Istanbul, 2Aksaray Hospital, Ophthalmology Clinics, Konya, Turkey Abstract: We report a case of unilateral transient mydriasis and ptosis after botulinum toxin injection applied by a medical doctor for a cosmetic procedure. A 36-year-old nurse was referred to our eye clinic with unilateral mydriasis and ptosis in the right eye 3 days after botulinum toxin injection for a cosmetic procedure. Botulinum toxin was applied to her eye by a doctor at her hospital who was not an ophthalmologist. She was treated with topical apraclonidine 0.5% ophthalmic solution. Her ptosis decreased to 2 mm with apraclonidine and her visual axis improved. Mydriasis was present for 3 weeks and then disappeared. Mild ptosis continued for 3 months, then resolved completely. Patients seeking treatment with botulinum toxin A for cosmetic purposes should be warned about the possibility of ptosis and mydriasis after injection. If these side effects are seen, the patient must be referred to an ophthalmologist for appropriate management. Keywords: botulinum toxin, mydriasis, ptosis

  10. The innovative therapeutic application of botulinum toxin type A in urology patients

    Directory of Open Access Journals (Sweden)

    Chrysoula Belai

    2016-06-01

    Full Text Available In the history of medical science the use of botulinum toxin was impressive. In the early 18th century it was defined as the neurotoxin implicated in the deadly disease botulism. Today, despite the toxic action finds application in the treatment of various diseases in a wide range of Medicine. Its use in urology was revolutionary in the treatment of neurogenic bladder, refractory idiopathic detrusor overactivity and other painful syndromes. The purpose of this review was to describe the treatment option of intravesical injection of botulinum toxin, in diseases of the urinary tract. The review showed that after many test applications under the experimental studies, the botulinum toxin type A has already established itself as the new treatment of choice after failure of conservative drug dealing in patients with neuro-urological symptoms of lower urinary tract. Cases of application of botulinum toxin in Urology are related to overactive bladder, neurogenic or idiopathic etiology, as bladder pain syndrome and chronic pelvic pain syndrome. According to the guidelines of the European Union directives Urology, the intravesical botulinum toxin injections are the most effective, minimally invasive treatment which results in reducing neurogenic hyperactivity of detrusor. In conclusion, this is a safe, easy and effective method that can be applied by health professionals, helping improve patients’ quality of life with neuro-urological diseases.

  11. Dynamic Model of Applied Facial Anatomy with Emphasis on Teaching of Botulinum Toxin A.

    Science.gov (United States)

    Boggio, Ricardo Frota

    2017-11-01

    The use of botulinum toxin type A is considered one of the most revolutionary and promising face rejuvenation methods. Although rare, most of the complications secondary to the use of botulinum toxin A are technician dependent. Among the major shortcomings identified in the toxin administration education is unfamiliarity with applied anatomy. This article proposes the use of body painting as an innovative method of teaching the application of botulinum toxin A. Using the body painting technique, facial anatomy was represented on the face of a model showing the major muscle groups of botulinum toxin A targets. Photographic records and films were made for documentation of represented muscles at rest and contraction. Using the body painting technique, each of the muscles involved in facial expression and generation of hyperkinetic wrinkles can be faithfully reproduced on the model's face. The documentation of the exact position of the points of application, the distribution of the feature points in the muscular area, the proper angulation and syringe grip, as well as the correlation of the points of application with the presence of hyperkinetic wrinkles, could be properly registered, providing professional training with information of great practical importance, development of highly effective treatments, and low complication rates. By making it possible to interrelate anatomy of a function, body painting is proposed in the present study as an innovative method, which in a demonstrative and highly didactic manner presents great potential as a teaching tool in the application of botulinum toxin A.

  12. A pilot study to evaluate effectiveness of botulinum toxin in treatment of androgenetic alopecia in males

    Directory of Open Access Journals (Sweden)

    Sehdev Singh

    2017-01-01

    Full Text Available Introduction: Androgenetic alopecia is a common form of alopecia with multifactorial etiology. Finasteride and minoxidil are approved by the FDA for the treatment of androgenetic alopecia. Balding scalp is believed to have relative microvascular insufficiency. Blood vessels in the scalp travel through the intramuscular plane. Intramuscular injection of botulinum toxin relaxes muscles and thereby increases blood flow in balding scalp. We conducted a pilot study to evaluate the efficacy of botulinum toxin in androgenetic alopecia management. Material and Methods: The study was conducted in a tertiary care center. A total of 10 male patients with androgenetic alopecia meeting inclusion criteria of the study were included. In the scalp, 30 sites were injected with 5 U of botulinum toxin in each site. Preprocedure photograph taken and evaluation was done, which was repeated after 24 weeks. Efficacy was assessed by photography and self-assessment scoring was done by patients. Results: Of 10 patients, 8 had good to excellent response on photographic assessment. At the end of 24 weeks, 1 patient showed poor and 1 showed fair response to treatment. As per self-assessment, 7of 10 patients showed good to excellent response. Two patients had fair response and 1 patient showed poor response to treatment. Conclusion: Botulinum toxin was found to be safe and effective therapy for the management of androgenetic alopecia in this pilot study. Studies with larger sample size and randomized controlled trials are required to establish the role of botulinum toxin in the management of androgenetic alopecia.

  13. Crystal structure of the receptor binding domain of the botulinum C-D mosaic neurotoxin reveals potential roles of lysines 1118 and 1136 in membrane interactions

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Buchko, Garry W.; Qin, Ling; Robinson, Howard; Varnum, Susan M.

    2011-01-07

    The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C-D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C (~two-thirds) and BoNT/D (~one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 Å resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal β-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR

  14. Epitope characterization and variable region sequence of f1-40, a high-affinity monoclonal antibody to botulinum neurotoxin type a (Hall strain.

    Directory of Open Access Journals (Sweden)

    Miles C Scotcher

    Full Text Available BACKGROUND: Botulism, an often fatal neuroparalytic disease, is caused by botulinum neurotoxins (BoNT which consist of a family of seven serotypes (A-H produced by the anaerobic bacterium Clostridium botulinum. BoNT, considered the most potent biological toxin known, is a 150 kDa protein consisting of a 100 kDa heavy-chain (Hc and a 50 kDa light-chain (Lc. F1-40 is a mouse-derived, IgG1 monoclonal antibody that binds the light chain of BoNT serotype A (BoNT/A and is used in a sensitive immunoassay for toxin detection. We report the fine epitope mapping of F1-40 and the deduced amino acid sequence of the variable regions of the heavy and light chains of the antibody. METHODS AND FINDINGS: To characterize the binding epitope of F1-40, three complementary experimental approaches were selected. Firstly, recombinant peptide fragments of BoNT/A light-chain were used in Western blots to identify the epitope domains. Secondly, a peptide phage-display library was used to identify the specific amino acid sequences. Thirdly, the three-dimensional structure of BoNT/A was examined in silico, and the amino acid sequences determined from the phage-display studies were mapped onto the three-dimensional structure in order to visualize the epitope. F1-40 was found to bind a peptide fragment of BoNT/A, designated L1-3, which spans from T125 to L200. The motif QPDRS was identified by phage-display, and was mapped to a region within L1-3. When the three amino acids Q138, P139 and D140 were all mutated to glycine, binding of F1-40 to the recombinant BoNT/A light chain peptide was abolished. Q-138, P-139 and D-140 form a loop on the external surface of BoNT/A, exposed to solvent and accessible to F1-40 binding. CONCLUSIONS: The epitope of F1-40 was localized to a single exposed loop (ss4, ss5 on the Lc of BoNT. Furthermore amino acids Q138, P139 and D140 forming the tip of the loop appear critical for binding.

  15. Structural Insights into the Functional Role of the Hcn Sub-domain of the Receptor-Binding Domain of the Botulinum Neurotoxin Mosaic Serotype C/D

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Gardberg, Anna; Edwards, Tom E.; Sankaran, Banumathi; Robinson, Howard; Varnum, Susan M.; Buchko, Garry W.

    2013-07-01

    Botulinum neurotoxin (BoNT), the causative agent of the deadly neuroparalytic disease botulism, is the most poisonous protein known for humans. Produced by different strains of the anaerobic bacterium Clostridium botulinum, BoNT effects cellular intoxication via a multistep mechanism executed by the three modules of the activated protein. Endocytosis, the first step of cellular intoxication, is triggered by the ~50 kDa, heavy-chain receptor-binding module (HCR) that is specific for a ganglioside and a protein receptor on neuronal cell surfaces. This dual receptor recognition mechanism between BoNT and the host cell’s membrane is well documented and occurs via specific intermolecular interactions with the C-terminal sub-domain, Hcc, of BoNT-HCR. The N-terminal sub-domain of BoNT-HCR, Hcn, comprises ~50% of BoNT-HCR and adopts a B-sheet jelly roll fold. While suspected in assisting cell surface recognition, no unambiguous function for the Hcn sub-domain in BoNT has been indentified. To obtain insights into the potential function of the Hcn sub-domain in BoNT, the first crystal structure of a BoNT with an organic ligand bound to the Hcn sub-domain has been obtained. Here, we describe the crystal structure of BoNT/CD-HCR determined at 1.70 Å resolution with a tetraethylene glycol (PG4) molecule bound in an hydrophobic cleft between B-strands in the B-sheet jelly fold roll of the Hcn sub-domain. The molecule is completely engulfed in the cleft, making numerous hydrophobic (Y932, S959, W966, and D1042) and hydrophilic (S935, W977, L979, N1013, and I1066) contacts with the protein’s side chain and backbone that may mimic in vivo interactions with the phospholipid membranes on neuronal cell surfaces. A sulfate ion was also observed bound to residues T1176, D1177, K1196, and R1243 in the Hcc sub-domain of BoNT/CD-HCR. In the crystal structure of a similar protein, BoNT/D-HCR, a sialic acid

  16. Crystal Structure of the Receptor Binding Domain of the botulinum C-D Mosiac Neurotoxin Reveals Potential Roles of Lysines 1118 and 1136 in Membrane Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Y Zhang; G Buchko; L Qin; H Robinson; S Varnum

    2011-12-31

    The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C-D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C ({approx}two-third) and BoNT/D ({approx}one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 {angstrom} resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal {beta}-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR.

  17. Content of botulinum neurotoxin in Botox®/Vistabel®, Dysport®/Azzalure®, and Xeomin®/Bocouture®.

    Science.gov (United States)

    Frevert, Jürgen

    2010-01-01

    Botulinum neurotoxin type A (BoNT/A) is the active substance in preparations used for the highly effective treatment of neurologic disorders such as cervical dystonia, blepharospasm, or spasticity, as well as other indications such as axillary and palmar hyperhidrosis, and urologic disorders. To determine the amount of BoNT/A protein present in pharmaceutical preparations of Botox®, Dysport®, and Xeomin®, which are identical with Vistabel®, Azzalure®, and Bocouture®, respectively. Rabbit and guinea pig antibodies raised against the 150 kD BoNT/A neurotoxin purified from Clostridium botulinum type A, strain ATCC 3502 ('Hall strain'), were used in a sensitive sandwich ELISA to determine the overall mean concentration of the 150 kD neurotoxin present in four batches of Botox® (C2344C3, C2384C3, C2419, and C2385), two batches of Dysport® (678F and 689X) and three batches of Xeomin® (61,111, 70,604, and 81,208). The specific neurotoxin potency, defined as the potency or biologic activity (units) per mass of neurotoxin protein (ng), was calculated based on the overall mean concentration of BoNT/A neurotoxin. Overall, the mean concentration of BoNT/A neurotoxin in Botox® was 0.73 ng per 100 unit vial (coefficient of variation [CV] = 3.5%), 3.24 ng per 500 unit vial of Dysport®, corresponding to 0.65 ng in 100 units (CV = 11.4%), and 0.44 ng per 100 unit vial of Xeomin® (CV = 1.9%). The specific potency of the 150 kD BoNT/A neurotoxin was calculated as 137 units/ng for Botox®, 154 units/ng Dysport®, and 227 units/ng Xeomin®. The current study has shown that of the three products, Xeomin® contains the highest specific neurotoxin activity, followed by Dysport®, with Botox® having the lowest specific activity. This result suggests that Xeomin® contains only active neurotoxin in contrast with Botox®, which is likely to contain additional denatured/inactive neurotoxin.

  18. Cryptic polyketide synthase genes in non-pathogenic Clostridium SPP.

    Directory of Open Access Journals (Sweden)

    Swantje Behnken

    Full Text Available Modular type I polyketide synthases (PKS produce a vast array of bacterial metabolites with highly diverse biological functions. Notably, all known polyketides were isolated from aerobic bacteria, and yet no example has been reported for strict anaerobes. In this study we explored the diversity and distribution of PKS genes in the genus Clostridium. In addition to comparative genomic analyses combined with predictions of modular type I polyketide synthase (PKS gene clusters in sequenced genomes of Clostridium spp., a representative selection of other species inhabiting a variety of ecological niches was investigated by PCR screening for PKS genes. Our data reveal that all studied pathogenic Clostridium spp. are devoid of putative PKS genes. In stark contrast, cryptic PKS genes are widespread in genomes of non-pathogenic Clostridium species. According to phylogenetic analyses, the Clostridium PKS genes have unusual and diverse origins. However, reverse transcription quantitative PCR demonstrates that these genes are silent under standard cultivation conditions, explaining why the related metabolites have been overlooked until now. This study presents clostridia as a putative source for novel bioactive polyketides.

  19. Botulinum toxin for treating muscular temporomandibular disorders: a systematic review

    Directory of Open Access Journals (Sweden)

    Eduardo Machado

    2012-12-01

    Full Text Available OBJECTIVE: This study, through a systematic literature review, aims to analyze the effectiveness of Botulinum Toxin as a treatment for masticatory myofascial pain and muscles temporomandibular disorders (TMD. METHODS: Survey in research bases: MEDLINE, Cochrane, EMBASE, Pubmed, Lilacs and BBO, between the years of 1966 and April 2011, with focus in randomized or quasi-randomized controlled clinical trials, blind or double-blind. RESULTS: After applying the inclusion criteria, 4 articles comprised the final sample: 3 were double-blind randomized controlled clinical trials and 1 was single-blind randomized controlled clinical trial. CONCLUSIONS: According to the literature, there is lack of evidence about the real effectiveness of botulinum toxin in the treatment of masticatory myofascial pain and muscular TMD. Thus, further randomized controlled clinical trials, with representative samples and longer follow-up time, to assess the real effectiveness of the technique are needed.OBJETIVO: este trabalho, por meio de uma revisão sistemática da literatura, teve como objetivo analisar a efetividade da toxina botulínica como tratamento para dor miofascial mastigatória e disfunções temporomandibulares (DTM musculares. MÉTODOS: pesquisa nas bases de dados Medline, Cochrane, Embase, Pubmed, Lilacs e BBO, no período entre 1966 e abril de 2011, com enfoque em estudos clínicos controlados randomizados ou quase-randomizados, cegos ou duplo-cegos. RESULTADOS: após a aplicação dos critérios de inclusão, chegou-se a 4 artigos, sendo que 3 eram estudos clínicos controlados randomizados duplo-cego e 1 era estudo clínico controlado randomizado simples-cego. CONCLUSÕES: pela análise da literatura, verificou-se um número reduzido de evidências significativas sobre a real efetividade da toxina botulínica no tratamento da dor miofascial e de DTM musculares. Assim, são necessários novos estudos clínicos controlados randomizados, com amostras

  20. A prediction model for Clostridium difficile recurrence

    Directory of Open Access Journals (Sweden)

    Francis D. LaBarbera

    2015-02-01

    Full Text Available Background: Clostridium difficile infection (CDI is a growing problem in the community and hospital setting. Its incidence has been on the rise over the past two decades, and it is quickly becoming a major concern for the health care system. High rate of recurrence is one of the major hurdles in the successful treatment of C. difficile infection. There have been few studies that have looked at patterns of recurrence. The studies currently available have shown a number of risk factors associated with C. difficile recurrence (CDR; however, there is little consensus on the impact of most of the identified risk factors. Methods: Our study was a retrospective chart review of 198 patients diagnosed with CDI via Polymerase Chain Reaction (PCR from February 2009 to Jun 2013. In our study, we decided to use a machine learning algorithm called the Random Forest (RF to analyze all of the factors proposed to be associated with CDR. This model is capable of making predictions based on a large number of variables, and has outperformed numerous other models and statistical methods. Results: We came up with a model that was able to accurately predict the CDR with a sensitivity of 83.3%, specificity of 63.1%, and area under curve of 82.6%. Like other similar studies that have used the RF model, we also had very impressive results. Conclusions: We hope that in the future, machine learning algorithms, such as the RF, will see a wider application.