bacteriophages nature s solution against pathogens · •greek “for bacteria eater” •natural...
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Bacteriophages
Nature’s Solution against Pathogens
Dirk de Meester
Micreos Food Safety
April 4th 2014
CEMI 19 Institut Pasteur – 4 avril 2014
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Micreos
• Dutch company based in
Wageningen
• Leader in applied phage
technology
• Strong scientific base
• SALMONELEX™ & LISTEX™
approved processing aid
• Human therapeutics & other
targets in the pipeline
A’dam
Wageningen
The Hague Bilthoven
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History with strong scientific background
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• Greek “for bacteria eater”
• Natural enemy of bacteria
• Every 48 hours 50% of the bacterial population is killed by phages
• Most abundant micro-organisms on earth (>1030)
• Outnumber bacteria 10 to 1
• 107 – 109 phages can be found in 1 ml of seawater
• Common residents intestinal tract, food, water, etc
Phages
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• Usually 100 times smaller than bacteria
• Harmless to humans animals and plants
• Affect only bacteria
• ‘species-specific’, and mostly specific within a species
• Do not affect the properties of treated food
• Leave no ecological footprint (composed of proteins and DNA, eventual breakdown products: amino acids and nucleic acids)
Phages
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Lytic cycle
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The Usual Suspects
E-coli
Listeria
Campylobacter
Salmonella
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Listeria monocytogenes
• Ubiquitous (also in modern processing equipment)
• Hardy: grows at refrigeration temperatures (0-40C) and in low oxygen conditions (i.e. packaged food)
• Minimum pH is 4.4, salt tolerant, pasteurization will kill Listeria
• Pathogenic, causes listeriosis
• Variable incubation (up to 2 months after exposure)
• Zero-tolerance
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We have solutions
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Fish
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Cheese
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(RTE) Meat Products
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Bacteriocidal effects measurable within
hours
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Efficacy on stainless steel
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Bagged whole muscle products Antimicrobials are sprayed into the vacuum packages
before sealing. Vacuum-sealing of the packages distributes
the antimicrobial evenly around the product.
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Hot dogs
Spray into the vacuum packages before sealing.
Vacuum-sealing of the packages distributes the
antimicrobial evenly around the product.
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Hot dogs Or LISTEX™ is sprayed directly into a hopper.
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Sliced Products
Spraying integrated into slicing systems, synchronized with
blade movement. Additional antimicrobials are added to
each package to cover all surfaces of the stack.
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A cost effective LISTEX™
application on Hot dogs
One unit treats 7.360
lbs
Cost-in-use is around
a penny per pound
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LISTEX ON SMEARED MUNSTER
CHEESE BY NIZO (Dutch Institute for Dairy Research)
Initial contamination: ~20 cfu/cm2
a. frequent high dose b. single high dose c. frequent low dose d.
control
a. 6x107 pfu/cm2
b. 1x107 pfu/cm2
c. 3,5x106 pfu/cm2
d. control
a b c d
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Ripening period is the problem
Cross-contamination during
smearing
Low incidence - high numbers in the
final product
Mostly surface ripened cheeses
SMEARED CHEESE
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Washing,
grading,
scaling
Injection
Dry salting
(24 h)
Washing
Drying
Smoking
Cutting
Dividing
Packaging
1
2
3
Washing,
Filleting
Dry salting
Drying
Smoking
Cutting
Dividing
Packaging
1
2
4
3
Washing,
grading,
scaling
Injection
Dry salting
(24 h)
Washing
Drying
Smoking
Cutting
Dividing
Packaging
1
2
3
Washing,
Filleting
Dry salting
Drying
Smoking
Cutting
Dividing
Packaging
1
2
4
3
Situation: Farmed fish is often contaminated Cold smoking and salting will not fully
remove Listeria Application:
Raw fillets: prevents transfer in facilities Smoked fish: kills after smoking Dipping or spraying
LISTEX™ controls Listeria in fish
processing
4b
4a
5a
5b
6
Adjustable
Adjustable
Adjustable
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Listeria occurance in a UK Salmon
plant
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LISTEX™ Regulatory Approvals
2006 FDA: GRAS for cheese products
2007 FDA: GRAS for all products
2010 The Netherlands Ministry of Health:
Processing aid
2011 Health Canada: processing aid
2011 USDA: processing Aid
2011 EFSA: LISTEX™ is safe for use
2012 FSANZ (Aus/NZ): processing aid
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Salmonelex™
Consists of a watery solution containing two
Salmonella-specific bacteriophages, Fo1a and
S16, which are produced and purified separately
and mixed in equal concentrations.
The commercial product has a minimal titer of
2x1011 pfu/mL.
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Host range
The two phages (S16 & FO1a) utilize different receptor
molecules. Testing of >200 Salmonella strains did not reveal
any strains that were not killed.
Felix O1 like (FO1a) phages utilize different receptor
molecules than S16, recognizing the terminal N-
acetylglucosamine residue of the outer LPS
(lipopolysaccharide) core.
S16 specifically recognizes the Salmonella outer membrane
protein C (ompC) which allows it to infect strains that have
rough or deep rough mutations, thus not requiring intact LPS
structure.
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Salmonella in the USA
• Salmonella infection has not declined in 15 years
• Performance standards set by USDA in July 2011 for poultry: <7,5% post-chill
• Salmonella infection accounts for $365 million in direct medical costs each year.
E. Coli O157
Salmonella
1998 2004
2010
Rela
tive r
ate
(Log s
cale
) 2.00
1.00
0.50
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Growout
&
Transport
Live hang Immobili-
zation Bleed out
Scalding Picking Eviscerati
on
Immersio
n &
chilling
Sorting &
Aging
Processe
d
Products
Packing
Poultry Processing Steps
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SALMONELEX™ Applications
• Chiller applications (pre-chiller)
• Trim prior to grinding
• Parts pre-packaging
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Interesting Opportunities in Pre-chiller
Applications
•Whole carcass rinse sampling methods show good
results post-chill however salmonella prevalence is
significantly higher after grinding and parts cut-up.
•Due to high organic load in the pre-chiller, chemicals
are ineffective and omitted in red water. Salmonelex™
however does not react away and prevents follicle
contamination and entrapment in the skin as the skin
shrinks and closes in the chiller.
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Pork
97,44
97,01
96,59
98,30
98,27
98,43
50 60 70 80 90 100
t=24h
t=48h
% reduction
con
tact
tim
e 1x10^7 pfu/cm²
2x10^7 pfu/cm²
91,87
92,36
93,49
94,97
95,00
95,84
50 60 70 80 90 100
t=24h
t=48h
t=6d
% reduction
con
tact
tim
e 1x10^7 pfu/cm²
2x10^7 pfu/cm²
Chicken Skin
92,11
91,72
91,70
97,39
96,41
97,01
50 60 70 80 90 100
t=24h
t=48h
% reduction
con
tact
tim
e 1x10^7 pfu/cm²
2x10^7 pfu/cm²
Chicken Breast
Fillet
Beef
Efficacy of SALMONELEX™
on Meat & Poultry
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The Benefits
• Leads to 1-3 log10 initial reductions
• Prevents cross contamination
• No organoleptic influence
• Does not dissolve fat
• Does not react away in the presence of organic matter
• No trade off between quality and safety
• Non corrosive
• Suitable for natural & organic products