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Knutsson R.,SVA National Veterinary Institute | van Rotterdam B.,RIVM National Institute for Public Health and the Environment | Fach P.,AFSSA French Food Safety Agency | De Medici D.,National Reference Center for Botulism | And 10 more authors.
International Journal of Food Microbiology | Year: 2011

A next frontier of the global food safety agenda has to consider a broad spectrum of bio-risks, such as accidental and intentional contaminations in the food and feed chain. In this article, the background for the research needs related to biotraceability and response to bioterrorism incidents are outlined. Given the current scale of international trade any response need to be considered in an international context. Biotraceability (e.g. the ability to use downstream information to point to processes or within a particular food chain that can be identified as the source of undesirable agents) is crucial in any food-born outbreak and particular in the response to bioterrorism events. In the later case, tested and proven biotraceability improves the following: (i) international collaboration of validated tracing tools and detection methods, (ii) multi-disciplinary expertise and collaboration in the field of food microbiology and conceptual modeling of the food chain, (iii) sampling as a key step in biotracing (iv) optimized sample preparation procedures, including laboratory work in Biosafety level 3 (BSL-3) laboratories, (v) biomarker discovery for relevant tracing and tracking applications, and (vi) high-throughput sequencing using bio-informatic platforms to speed up the characterization of the biological agent. By applying biotraceability, the response phase during a bioterrorism event may be shortened and is facilitated for tracing the origin of biological agent contamination. © 2010 Elsevier B.V.

Segerman B.,National Veterinary Institute SVA | De Medici D.,University of Veterinary Medicine Vienna | Ehling Schulz M.,National Reference Center for Botulism | Fach P.,AFSSA French Food Safety Agency | And 5 more authors.
International Journal of Food Microbiology | Year: 2011

The rapid technological development in the field of parallel sequencing offers new opportunities when tracing and tracking microorganisms in the food and feed chain. If a bioterror organism is deliberately spread it is of crucial importance to get as much information as possible regarding the strain as fast as possible to aid the decision process and select suitable controls, tracing and tracking tools. A lot of efforts have been made to sequence multiple strains of potential bioterror organisms so there is a relatively large set of reference genomes available. This study is focused on how to use parallel sequencing for rapid phylogenomic analysis and screen for genetic modifications. A bioinformatic methodology has been developed to rapidly analyze sequence data with minimal post-processing. Instead of assembling the genome, defining genes, defining orthologous relations and calculating distances, the present method can achieve a similar high resolution directly from the raw sequence data. The method defines orthologous sequence reads instead of orthologous genes and the average similarity of the core genome (ASC) is calculated. The sequence reads from the core and from the non-conserved genomic regions can also be separated for further analysis. Finally, the comparison algorithm is used to visualize the phylogenomic diversity of the bacterial bioterror organisms Bacillus anthracis and Clostridium botulinum using heat plot diagrams. © 2010 Elsevier B.V.

Woudstra C.,ANSES French Agency for Food | Lambert D.,Botulism Reference Service for Canada | Anniballi F.,National Reference Center for Botulism | De Medici D.,National Reference Center for Botulism | And 2 more authors.
Applied and Environmental Microbiology | Year: 2013

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 presentstudy 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 typeswith certain geographical areas make gene profiling of flaVR and flaB promising in molecular surveillance and epidemiology of C. botulinum. © 2013, American Society for Microbiology.

Fillo S.,Army Medical and Veterinary Research Center | Giordani F.,Army Medical and Veterinary Research Center | Anniballi F.,National Reference Center for Botulism | Gorge O.,Directorate General of Armaments | And 11 more authors.
Journal of Clinical Microbiology | Year: 2011

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 C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Giordani F.,Army Medical and Veterinary Research Center | Fillo S.,Army Medical and Veterinary Research Center | Anselmo A.,Army Medical and Veterinary Research Center | Palozzi A.M.,Army Medical and Veterinary Research Center | And 11 more authors.
Infection, Genetics and Evolution | Year: 2015

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. © 2015 Elsevier B.V.

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