fabrizio anniballi alfonsina fiore, bruna auricchio, dario...

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

[email protected] - [email protected]

mailto:[email protected]



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”



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



MECHANISM OF ACTION

Binding Endocytosis Translocation (low pH) Hydrolysis of SNARE proteins



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



CURRENT TREATMENT

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



BoNT-PRODUCING CLOSTRIDIA



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



Inhibition of spore germination, growth, toxinogenesis

Acidification Brine Use of preservatives

Destruction of spores

MEASURES AGAINST BoNT-PRODUCING CLOSTRIDIA



Nowadays changed behaviors among people Fresh-like products Hurdles technologies

Bio-competition



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

THE PROJECT



Collaborative project funded by the Ministry of Defence

Dr FLORIGIO LISTA

Dr DARIO DE MEDICI Prof CESARE MONTECUCCO

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

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.




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.




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.




The future perspective of this projects is the development of a clinical trial among human volunteers.




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




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




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)




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

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

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

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

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)

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

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.



Thank you for your attention !

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