Closing Clostridium botulinum Group III Genomes Using Long-Read Sequencing

Clostridium botulinum group III is the anaerobic Gram-positive bacterium producing the deadly neurotoxin responsible for animal botulism. Here, we used long-read sequencing to produce four complete genomes from Clostridium botulinum group III neurotoxin types C, D, C/D, and D/C. The protocol for obtaining high-molecular-weight DNA from C. botulinum group III is described.Peer reviewe

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

<p>Abstract</p> <p>Background</p> <p><it>Clostridium botulinum </it>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 <it>C. botulinum </it>strain from physiological group III, causing animal botulism. We also compare the sequence to three new draft genomes from the same physiological group.</p> <p>Results</p> <p>The 2.77 Mb chromosome was highly conserved between the isolates and also closely related to that of <it>C. novyi</it>. However, the sequence was very different from the human <it>C. botulinum </it>group genomes. Replication-directed translocations were rare and conservation of synteny was high. The largest difference between <it>C. botulinum </it>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.</p> <p>Conclusions</p> <p>The completion of the genome of <it>C. botulinum </it>group III has revealed it to be a genome with dual identity. It belongs to the pathogenic species <it>C. botulinum</it>, but as a genotypic species it should also include <it>C. novyi </it>and <it>C. haemolyticum</it>. The genotypic species share a conserved chromosomal core that can be transformed into various pathogenic variants by modulation of the highly plastic plasmidome.</p

The Workshop on Animal Botulism in Europe

A workshop on animal botulism was held in Uppsala, Sweden, in June 2012. Its purpose was to explore the current status of the disease in Europe by gathering the European experts in animal botulism and to raise awareness of the disease among veterinarians and others involved in biopreparedness. Animal botulism is underreported and underdiagnosed, but an increasing number of reports, as well as the information gathered from this workshop, show that it is an emerging problem in Europe. The workshop was divided into 4 sessions: animal botulism in Europe, the bacteria behind the disease, detection and diagnostics, and European collaboration and surveillance. An electronic survey was conducted before the workshop to identify the 3 most needed discussion points, which were: prevention, preparedness and outbreak response; detection and diagnostics; and European collaboration and surveillance. The main conclusions drawn from these discussions were that there is an urgent need to replace the mouse bioassay for botulinum toxin detection with an in vitro test and that there is a need for a European network to function as a reference laboratory, which could also organize a European supply of botulinum antitoxin and vaccines. The foundation of such a network was discussed, and the proposals are presented here along with the outcome of discussions and a summary of the workshop itself

Management of Animal Botulism Outbreaks: From Clinical Suspicion to Practical Countermeasures to Prevent or Minimize Outbreaks

Botulism is a severe neuroparalytic disease that affects humans, all warm-blooded animals, and some fishes. The disease is caused by exposure to toxins produced by Clostridium botulinum and other botulinum toxin–producing clostridia. Botulism in animals represents a severe environmental and economic concern because of its high mortality rate. Moreover, meat or other products from affected animals entering the food chain may result in a public health problem. To this end, early diagnosis is crucial to define and apply appropriate veterinary public health measures. Clinical diagnosis is based on clinical findings eliminating other causes of neuromuscular disorders and on the absence of internal lesions observed during postmortem examination. Since clinical signs alone are often insufficient to make a definitive diagnosis, laboratory confirmation is required. Botulinum antitoxin administration and supportive therapies are used to treat sick animals. Once the diagnosis has been made, euthanasia is frequently advisable. Vaccine administration is subject to health authorities' permission, and it is restricted to a small number of animal species. Several measures can be adopted to prevent or minimize outbreaks. In this article we outline all phases of management of animal botulism outbreaks occurring in wet wild birds, poultry, cattle, horses, and fur farm animals

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

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

Mot en elektrifierad framtid : En studie om attityden till elbilar i Las Vegas

Syftet med den här studien är att undersöka vilka faktorer som styr olika generationers attityd till elbilen i staden Las Vegas i USA och hur dessa attityder kan forma ett potentiellt köpbeteende. Generationerna som undersöktes var Gen X, Y och Z. Mätningen av attityd i studien inspirerades från Tricomponent Attitude Model, som mäter konsumenternas kognitiva, affektiva och konativa attityd. Studien använde sig av en enkätundersökning med påståenden för att fånga attityder hos konsumenter i staden, vilket gjorde att uppsatsen antog sig en kvalitativ forskningsmetod. Enkäten fick in 64 svar som sedan analyserades utifrån attitydkomponenterna i relation till faktorer, som gav undersökningen numerära verktyg att använda vid mätning av potentiellt köpbeteende. Resultatet visade att Gen X hade ett negativt potentiellt köpbeteende, medan Gen Y och Z hade ett positivt potentiellt köpbeteende. Analysen utgick från att hitta ledtrådar i generationernas grundattityder till de presenterade faktorerna och som därmed kunde förklara köpbeteendet

Horizontal gene transfer of toxin genes in Clostridium botulinum: Involvement of mobile elements and plasmids

Intoxication with the potent botulinum neurotoxin (BoNT) gives rise to the serious paralytic illness botulism. BoNT is part of a complex that consists of the neurotoxin and several associated components, all encoded by the bont gene cluster. This gene cluster has likely been subjected to horizontal gene transfer between different groups of clostridia, which has given rise to the genetically diverse species Clostridium botulinum. C. botulinum is divided into four physiological groups (I–IV), where group I and II cause disease in humans and group III in animals. Analysis of the genomes of group I, II and III has revealed that toxin genes, including the bont cluster, often are plasmid-borne. The genomes analyzed from group III contain an unusually high number of plasmids carrying different toxin genes. Some of these genes are also found in other Clostridium species and some have moved between different plasmids within the same physiological group. This indicates that horizontal transfer of toxin genes is taking place within and between species of Clostridium. The abundance of mobile elements, especially in genomes of group III, is likely connected to accelerated genome plasticity and gene transfer events

Sequence comparison between the C-St BoNT-encoding phage and the p1Ch9693 phage from a <i>C. haemolyticum</i> strain.

<p>Artemis Comparison Tool (ACT) plot of an alignment between the C-St BoNT-encoding phage from <i>C. botulinum</i> strain C-Stockholm and p1Ch9693 from <i>C. haemolyticum</i> strain NCTC 9693. Regions of similarity are indicated in red (same direction) or blue (opposite direction). Genes in the <i>bont</i> cluster are green, genes homologous between the two replicons are orange, and remaining genes are gray.</p

Average whole genome similarity of <i>C. novyi sensu lato</i> genomes.

<p>A similarity matrix based on normalized average BLASTN sores for fragmented comparisons covering the whole genomes (200 bp fragment size) illustrated by a heat-plot. Four lineages were identified (I–IV) and are framed with black squares. The geographical origins of the strains are listed with two-digit country codes. Strains isolated more than 50 years ago are marked with a star.</p

Comparative analysis of <i>C. novyi sensu lato</i> plasmids.

<p>A heat-plot of the relative amount of shared genetic material between plasmids (40% normalized BLASTN score threshold). Thirteen plasmid groups (PG1–PG13), framed by black squares, were identified from these results and from analysis of plasmid replication and partitioning genes. Some of the plasmids in lineage I strains (PG1–PG3) shared several IS elements, and this resulted in an apparent increase in the background level of shared genetic content between otherwise dissimilar plasmids within this lineage. Uncompleted plasmids are marked with a star.</p