Genomic organization and diversity of Clostridium

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Acta Universitatis agriculturae Sueciae, Cover Agar plate with colonies of a Clostridium botulinum group III strain. photo Karl Erik Johansson BVF The images of the DNA spiral and the. bacteriophage are obtained from Pixabay,ISSN 1652 6880. ISBN print version 978 91 576 8358 8,ISBN electronic version 978 91 576 8359 5. 2015 Hanna Skarin Uppsala,Print SLU Service Repro Uppsala 2015. Genomic organization and diversity of Clostridium botulinum. group III the bug behind animal botulism, 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 animals. relatively commonly affected are cattle horses sheep and farmed fur animals C. botulinum is a diverged species and can be organized into four groups which reflect. their genetic and physiological differences C botulinum group III strains producing. BoNT types C D and chimers C D and D C are mainly connected to animal botulism. The gene encoding BoNT in C botulinum group III strains is located on an unstable. plasmid like phage, In this thesis strains of the previously relatively uncharacterised C botulinum group. III were isolated and genotyped with pulsed field gel electrophoresis Several. pulsotypes were formed but the majority clustered closely together and represented. most of the chimeric strains Strains representing different pulsotypes and different. animal and geographical origin were selected for whole genome sequencing and the. resulting genomes could be divided into four genomic lineages Comparisons against. genomes of Clostridium novyi and Clostridium haemolyticum revealed that they could. be organized into the same genomic lineages lineages II IV which resulted in the. suggested collective term C novyi sensu lato The organization of all sequenced. genomes was analysed It revealed a relatively conserved chromosome and an. abundance of highly dynamic plasmids The plasmids lineages and species were. entwined because plasmids and toxin genes had moved across the lineage boundaries. Of the four lineages only lineage I was C botulinum specific and this lineage includes. strains of the most common pulsotype One genome of lineage I was assembled into. completion It was smaller than C botulinum group I and II genomes but contained as. much as five plasmids Most of the identified putative toxin genes were found on these. plasmids Strains of lineage I may be more virulent than other C botulinum group III. strains which is reflected by their domination in animal botulism cases today. Keywords Clostridium botulinum botulinum neurotoxin BoNT botulism PFGE. genome plasmid bacteriophage Clostridium novyi Clostridium haemolyticum. Author s address Hanna Skarin SVA Department of Bacteriology 756 51 Uppsala. Sweden E mail hanna skarin sva se,Till min morfar som r en riktig biolog. List of Publications 7,Additional related publications 9. Abbreviations 10,1 Foreword 11,2 Background 13,2 1 Clostridium botulinum and botulism 13. 2 1 1 A historical glance 13,2 1 2 Etiology 13,2 1 3 Taxonomy and classification 14.
2 1 4 The botulinum neurotoxin 15,2 1 5 The disease mechanism 16. 2 1 6 C botulinum and BoNT as a biothreat 18,2 2 Genomics 18. 2 2 1 Bacterial genome sequencing 18,2 2 2 C botulinum genomics 20. 2 2 3 Mobile genetic elements and horizontal gene transfer 22. 2 3 Animal Botulism 24,2 3 1 Disease routes 24,2 3 2 Avian botulism 25. 2 3 3 C botulinum group III virulence factors 26,2 3 4 Diagnostics 27.
3 Aims of the thesis 31,4 Comments on Materials and Methods 33. 4 1 Isolates 33,4 1 1 Sampling materials 33,4 1 2 Cultivation 33. 4 1 3 Isolation 34,4 1 4 Identification of strains 34. 4 2 DNA extraction 36,4 3 Genotyping 36,4 4 Sequencing and assembly 37. 4 4 1 Sequencing technology 37,4 4 2 Genome assembly 37.
4 4 3 Plasmid assembly 38,4 4 4 Annotation 38,4 5 Genome comparative analysis 38. 4 6 Plasmid analysis 39,5 Results and Discussion 41. 5 1 Collection and identification of strains paper I II and IV 41. 5 2 Genomic diversity in C botulinum group III paper I II III and IV 42. 5 3 The C botulinum group III genome paper III 44,5 4 C novyi sensu lato paper I III and IV 46. 5 5 The large mobile plasmidome of C novyi sensu lato paper III and IV 47. 5 6 Pathogenicity in C novyi sensu lato paper III and IV 49. 5 7 The successful lineage I 52,6 Conclusions 53,7 Perspectives for the future 55. 8 Popul rvetenskaplig sammanfattning 57,9 Acknowledgements 61.
10 References 64,List of Publications, This thesis is based on the work contained in the following papers referred to. by Roman numerals in the text, I Skarin H Lindberg A Blomqvist G Asp n A and B verud V. 2010 Molecular characterization and comparison of Clostridium. botulinum type C avian strains Avian Pathology 39 6 511 518. II Anza I1 Skarin H1 Vidal D Lindberg A B verud V and Mateo R. 2014 The same clade of Clostridium botulinum strains is causing avian. botulism in southern and northern Europe Anaerobe 26 20 23. 1These authors contributed equally to this publication. III Skarin H H fstr m T Westerberg J and Segerman B 2011. Clostridium botulinum group III a group with dual identity shaped by. plasmids phages and mobile elements BMC Genomics 12 185. doi 10 1186 1471 2154 12 185, IV Skarin H and Segerman B 2014 Plasmidome interchange between. Clostridium botulinum Clostridium novyi and Clostridium haemolyticum. converts strains of independent lineages into distinctly different pathogens. PLoS ONE 9 9 e107777 doi 10 1371 journal pone 0107777. Papers I and II are reproduced with the permission of the publisher. The contribution to the papers included in this thesis was as follows. I Hanna Skarin Anna Lindberg and Viveca B verud planned and designed. the experiments Hanna Skarin performed the experiments and analysed. the results together with Anna Asp n Hanna Skarin wrote the first draft of. the manuscript with input from Anna Lindberg Gunilla Blomqvist Viveca. B verud and Anna Asp n, II Hanna Skarin and Ibone Anza Gomez planned the study Hanna Skarin. performed the Immunomagnetic separation experiments Ibone Anza. Gomez collected samples from Spanish avian botulism outbreaks Hanna. Skarin and Ibone Anza Gomez performed isolation and genotyping. experiments and analysed the results Hanna Skarin and Ibone Anza Gomez. wrote the manuscript with input from Dolors Vidal Rafael Mateo Anna. Lindberg and Viveca B verud, III Hanna Skarin and Bo Segerman conceived and designed the experiments.
Hanna Skarin Therese H fstr m and Josefina Westerberg performed the. experiments Hanna Skarin Therese H fstr m and Bo Segerman analysed. the results Hanna Skarin and Bo Segerman wrote the manuscript. IV Hanna Skarin and Bo Segerman conceived and designed the experiments. Hanna Skarin performed the experiments Hanna Skarin and Bo Segerman. analysed the results and wrote the manuscript,Related publications. Additional publications have been produced during this thesis project which. also relates to the topic, Lindberg A Skarin H Knutsson R Blomqvist G and B verud V. 2010 Real time PCR for Clostridium botulinum type C neurotoxin. BoNTC gene also covering a chimeric C D sequence application on. outbreaks of botulism in poultry Veterinary Microbiology 146 1 2 118. Skarin H and Segerman B 2011 Horizontal transfer of toxin genes in. Clostridium botulinum Involvement of mobile elements and plasmids. Mobile Genetic Elements 1 3 213 215, Woudstra C Skarin H Anniballi F Fenicia L Bano L Drigo I. Koene M B yon Auboyer M H Buffereau J P De Medici D and. Fach P 2012 Neurotoxin gene profiling of Clostridium botulinum types. C and D native to different countries within Europe Applied Environmental. Micriobiology 78 9 3120 3127, Skarin H Tevell berg A Woudstra C Hansen T L fstr m C. Koene M Bano L Hedeland M Anniballi F De Medici D Olsson. Engvall E 2013 The workshop on animal botulism in Europe. Biosecurity and bioterrorism biodefense strategy practice and science. 11 Suppl 1 183 190, Skarin H Lindgren Y and Jansson D 2015 Investigations into an.
outbreak of botulism caused by Clostridium botulinum type C D in laying. hens Avian Diseases 59 335 340,Abbreviations,AFLP amplified fragment length polymorphism. BoNT botulinum neurotoxin,bont botulinum neurotoxin gene. bp base pair,contig contiguous sequence,DURC dual use research of concern. HA hemagglutinin,HC heavy chain,LC light chain,MLST multilocus sequence typing. MLVA multiple locus variable number tandem repeat analysis. NAP non toxic neurotoxin associated proteins,NGS next generation sequencing.
NTNH non toxic non hemagglutinin protein,PCR polymerase chain reaction. PFGE pulsed field gel electrophoresis,RAPD randomly amplified polymorphic DNA analysis. SNARE soluble N ethylmaleimide sensitive factor attachment protein. SVA Swedish National Veterinary Institute,TeTx Clostridium tetani toxin. VAMP vesicle associated membrane protein,1 Foreword. F geld den kan sprida livsfarlig sjukdom,Transl Bird deaths can spread deadly disease.
Those words were the heading of an article hanging on the wall in the. laboratory where I started studying botulism It was in the midst of the tumult. caused by the increasing number of avian botulism outbreaks in Sweden. Along with the outbreaks there was a flow of samples coming into the. laboratory for analysis All day long I cut pieces of chicken caeca cultivated. them and analysed them for the presence of Clostridium botulinum It was a. tedious job not to mention the very badly smelling working material However. I did not know it at the time but these foul smelling chicken samples later. became the foundation of this thesis The number of reported animal botulism. outbreaks has increased during the last decade not only in Sweden but in. several European countries Skarin et al 2013 As C botulinum produces an. extremely toxic neurotoxin botulism research became a part of an EU project. AniBioThreat which aimed to improve the EU s capacity to counter biological. animal bio threats in terms of awareness prevention and contingency. AniBioThreat 2010 This opened up the possibility for a PhD project about. botulism Now after four years of research this thesis represents the content of. that PhD project,2 Background,2 1 Clostridium botulinum and botulism. 2 1 1 A historical glance, The first mentioning of botulinum poisoning was in Germany in the 17th. century where patients who had eaten blood sausages suffered from paralytic. symptoms The word botulism comes from the Latin word for sausage botulus. However the bacterium itself was not mentioned until 1897 in Belgium where. several people became ill and died from eating incompletely salted ham at a. wake A professor in Ghent named mile van Ermengem examined the ham in. a microscope and discovered rod shaped bacteria He called them Bacillus. botulinus having in mind the previous reports from Germany where similar. symptoms had been described Van Ermengem established that the illness of. botulism resulted from an extremely potent toxin that was produced by this. bacterium van Ermengem 1979 In 1917 the genus Bacillus was divided into. Bacillus for aerobic species and Clostridium for anaerobic species thus. Bacillus botulinus was changed into Clostridium botulinum Winslow et al. 2 1 2 Etiology, Clostridium botulinum is a Gram positive anaerobic rod shaped bacterium. which occurs as single rods or in short chains Figure 1 The rods measure. 0 3 1 9 um in width and 1 6 9 4 um in length Smith and Hobbs 1974 They. have flagella and form oval endospores with a varying resistance to heat from. very high to low depending on the strain Table 2 Ito et al 1967 Segner. and Schmidt 1971 Peck et al 2011 These spores are distributed in soil and. aquatic sediments around the world Hauschild 1989 Hatheway 1990 C. botulinum produces extremely potent botulinum neurotoxins BoNTs. probably with the purpose to kill the host by intoxication It can propagate. from nutrients made available by saprophytic organisms and produce spores. and toxin in the anaerobic environment provided by the dead host thereby. ensuring its own spread through larvae or scavenging animals BoNTs cause. the disease botulism affecting both humans and animals although it is more. common in animals Critchley 1991 The mortality is high if treatment with. antitoxins and respiratory support is not given WHO 2013 Animals which. are seldom treated are occasionally victims to large botulism outbreaks. Figure 1 Scanning electron microscopy image of C botulinum vegetative cells Photo H Skarin. SVA and Leif Ljung Uppsala University,2 1 3 Taxonomy and classification. C botulinum is a taxonomic designation for bacteria that produce one or. more BoNTs Smith and Hobbs 1974 In the beginning of the 20th century it. was noted that different strains of C botulinum produced toxins that had. different antigenicities since antitoxins raised towards one toxin did not. Genomic organization and diversity of Clostridium botulinum group III The bug behind animal botulism Hanna Skarin Faculty of Veterinary Medicine and Animal Science Department of Biomedical Sciences and Veterinary Public Health Uppsala and Department of Bacteriology National Veterinary Institute Uppsala Doctoral Thesis Swedish University of Agricultural Sciences Uppsala 2015 Acta

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