Fabrizio Anniballi Alfonsina Fiore, Bruna Auricchio, Dario De Medici

Transcription

1 Fabrizio Anniballi Alfonsina Fiore, Bruna Auricchio, Dario De Medici Istituto Superiore di Sanità Department of Veterinary Public Health and Food Safety Unit of Microbiological Hazards National Reference Centre for Botulism Viale Regina Elena, Roma Phone Fax cnr.botulismo@iss.it

2 OUTLINE Introduction Botulinum toxins - structure Botulinum toxins- mechanism of action BoNT-producing clostridia Measures for botulism prevention and therapy Forms of botulism Findings of New Inhibitors of Botulism Findings of Setting up of Bio-competition and Decontamination Methods to Improve Technological Processes and Food Safety

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4 BoNTs BoNTs are the most potent poisons yet known Classified by the CDC as Tier One select agents Human therapeutics against several neuromuscular disorders The high potency depends on the enzymatic activity and on the selective affinity for binding neurons 7 types confirmed, 1 new type, 40 sub-types

5 MECHANISM OF ACTION Binding Endocytosis Translocation (low ph) Hydrolysis of SNARE proteins

6 MEASURES AGAINST BOTULISM Vaccination Undesirable because the rarity of botulism Preclude the use of BoNTs for their beneficial effects Some soldiers in USA are vaccinates

7 CURRENT TREATMENT Artificial respiration Passive immunization Act only on circulating toxins First days after onset symptoms Adverse effects

8 BoNT-PRODUCING CLOSTRIDIA Clostridium baratii tox type F Clostridium botulinum tox types A-B-C-D-E-F-G-H Clostridium butyricum tox type E anaerobes, spore-forming, ubiquitous, different phenotypic and genotypic features

9 MEASURES AGAINST BoNT-PRODUCING CLOSTRIDIA Inhibition of spore germination, growth, toxinogenesis Acidification Brine Use of preservatives Destruction of spores

10 MEASURES AGAINST BoNT-PRODUCING CLOSTRIDIA Nowadays changed behaviors among people Fresh-like products Hurdles technologies Bio-competition

11 FORMS of BOTULISM Toxin pre-formed in foods Un-correct use of toxin Toxin produced in vivo in intestinal lumen of infants Toxin produced in vivo in intestinal lumen of adults Toxin produced in vivo in a wound

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13 THE PROJECT Collaborative project funded by the Ministry of Defence Dr FLORIGIO LISTA Dr DARIO DE MEDICI Prof CESARE MONTECUCCO

14 THE MAIN OBJECTIVES 1 Identification of some new inhibitors of BoNTs. Carried out by the University of Padua 2 Characterization of BoNT-producing clostridia through WGS. Carried out by NRCB and Army Medical Research Centre

15 MAIN FINDINGS OBJECTIVE 1

16 MAIN FINDINGS OBJECTIVE 1 Thioredoxin reductase-thioredoxin is highly expressed in the motor neurons terminals and it is present on the synaptic vesicles cytosolic surface. The inhibition of this redox system can prevent the SNARE protein cleavage.

17 MAIN FINDINGS OBJECTIVE 1 This redox system is implicated in controlling a variety of cell functions altered in a number of diseases. Several drugs have been identified: anaurofin, curcumin, myricetin, juglone, ebselen, px-12. All of these molecules protect against BoNT/A, /E, C in vitro and also in vivo models.

18 MAIN FINDINGS OBJECTIVE 1 Most of these substances are nontoxic and included in clinical trial for other diseases. Although these substance cannot reverse the paralysis, they may be use immediately after clinical diagnosis particularly in infant botulism cases. The use of these substances avoid the adverse effect of passive immunization with sera.

19 MAIN FINDINGS OBJECTIVE 1 The future perspective of this projects is the development of a clinical trial among human volunteers.

20 MAIN FINDINGS OBJECTIVE 2 Characterization of BoNT producing clostridia strains. 350 tested strains were chosen among CNRB strain collection (more than 600 strains). They were representative of all forms of botulisms, all Italian Regions and isolated during the period Screened with MLVA

21 MAIN FINDINGS OBJECTIVE 2 Ten main clusters were identified At least one representative strain for each cluster were submitted to WGS using both Roche 454 and Illumina MiSeq platforms 2 paper published and an additional paper submitted to Applied and Environmental Microbiology

22 MAIN FINDINGS OBJECTIVE 2

23 MAIN FINDINGS OBJECTIVE 2 Ten strains sequenced In 6 genomes 1 or 2 plasmids were found BoNT gene was carried out in plasmid 4 strains In the genome BoNT gene is located in oppa/brnq operon (4 type B strains) and in arsc operon (2 strains)

24 MAIN FINDINGS OBJECTIVE 2 Clonal phylogeny conducted on Italian strains and the other strains fully sequenced shows that Italian strains do not form a monophyletic unit (high variability of these strains) A new sub-type of BoNT/F was detected

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26 THE PROJECT Collaborative project funded by the Ministry of Health 10 Italian Institutes Dr ALFONSINA FIORE Bio-competition LAB vs BoNT-producing clostridia LAB vs Cronobacter sakazakii

27 THE MAIN OBJECTIVE Evaluate bio-competition among Lactic-Acid-Bacteria and BoNT-producing clostridia with the aim of using LAB and/or bacteriocins as bio-preservative against botulism

28 MATERIAL AND METHODS Antimicrobial activity of: 6 strains of Lactococcus lactis sup. lactis (nisin) 9 strains of Streptococcus thermophilus (thermophilin) 9 strains of Bacillus coagulans (nisin) was evaluated against 35 group I BoNT-producing clostridia strains mainly isolated from infant botulism cases Spot on the lawn method was chosen to carry out these activities

29 MAIN FINDINGS 100 % of tested BoNT-producing clostridia strains were sensitive to nisin and coagulin 86% were positive to thermophilin These results were confirmed detecting genes encoding for bacteriocins in LAB strains Results were submitted to BACTIBASE (the specific database dedicated to bacteriocins)

30 MATERIAL AND METHODS Antimicrobial activity of: 6 strains of Lactococcus lactis sup. lactis (nisin) 7 strains of Streptococcus thermophilus (thermophilin) 8 strains of Bacillus coagulans (nisin) 17 Bifidobacterium spp. was evaluated against 2 group III Clostridium botulinum (1 type CD and 1 type DC) strains isolated from poultry and cattle faeces of botulism-affected aninals

31 MAIN FINDINGS All Lactococcus lactis, Bacillus subtilis and Steptococcus thermophilus tested strains are capable of inhibiting group III C. botulinum strains Nisin showed the highest antimicrobial activity Of the 17 Bifidobacterium spp strains tested only Bifidobacterium bifidum ATCC35014 exerted inhibition of group III C. botulinum strains The use of feed containing probiotics could promote beneficial flora to compete with pathogens such us group III C. botulinum.

32 Thank you for your attention!