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Fabrizio Anniballi Alfonsina Fiore, Bruna Auricchio, Dario De Medici
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
Istituto Superiore di Sanità
Department of Veterinary Public Health and Food Safety
Unit of Microbiological Hazards
National Reference Centre for Botulism
Viale Regina Elena, 299 – 00161 Roma
Phone. +39 06 4990 2254 – Fax +39 06 4990 2045
fabrizio.anniballi@iss.it - cnr.botulismo@iss.it
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”
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
MECHANISM OF ACTION
Binding Endocytosis Translocation (low pH) Hydrolysis of SNARE proteins
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
CURRENT TREATMENT
Artificial respiration Passive immunization Act only on circulating toxins First days after onset symptoms Adverse effects
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
BoNT-PRODUCING CLOSTRIDIA
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
Clostridium butyricum tox type E
Clostridium baratii tox type F
Clostridium botulinum
tox types A-B-C-D-E-F-G-H
anaerobes, spore-forming, ubiquitous, different phenotypic and genotypic features
MEASURES AGAINST BoNT-PRODUCING CLOSTRIDIA
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
Inhibition of spore germination, growth, toxinogenesis
Acidification Brine Use of preservatives
Destruction of spores
MEASURES AGAINST BoNT-PRODUCING CLOSTRIDIA
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
Nowadays changed behaviors among people Fresh-like products Hurdles technologies
Bio-competition
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
FORMS of BOTULISM
Toxin pre-formed in foods
Toxin produced in vivo in intestinal lumen of adults
Toxin produced in vivo in intestinal lumen of infants
Toxin produced in vivo in a wound
Un-correct use of toxin
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
THE PROJECT
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
Collaborative project funded by the Ministry of Defence
Dr FLORIGIO LISTA
Dr DARIO DE MEDICI Prof CESARE MONTECUCCO
THE MAIN OBJECTIVES
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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
MAIN FINDINGS OBJECTIVE 1
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
MAIN FINDINGS OBJECTIVE 1
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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.
MAIN FINDINGS OBJECTIVE 1
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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.
MAIN FINDINGS OBJECTIVE 1
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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.
MAIN FINDINGS OBJECTIVE 1
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
The future perspective of this projects is the development of a clinical trial among human volunteers.
MAIN FINDINGS OBJECTIVE 2
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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 1984-2013
Screened with MLVA
MAIN FINDINGS OBJECTIVE 2
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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
MAIN FINDINGS OBJECTIVE 2
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
MAIN FINDINGS OBJECTIVE 2
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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)
MAIN FINDINGS OBJECTIVE 2
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
THE PROJECT
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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
THE MAIN OBJECTIVE
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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
MATERIAL AND METHODS
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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
MAIN FINDINGS
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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)
MATERIAL AND METHODS
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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
MAIN FINDINGS
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
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.
4 th FOODSEG SYMPOSIUM
23rd - 24th of April 2015 - Istituto Superiore di Sanità – Rome, ITALY
Thank you for your attention !
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