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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
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
History with strong scientific background
• 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
• 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
Lytic cycle
The Usual Suspects
E-coli
Listeria
Campylobacter
Salmonella
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
We have solutions
Fish
Cheese
(RTE) Meat Products
Bacteriocidal effects measurable within
hours
Efficacy on stainless steel
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.
Hot dogs
Spray into the vacuum packages before sealing.
Vacuum-sealing of the packages distributes the
antimicrobial evenly around the product.
Hot dogs Or LISTEX™ is sprayed directly into a hopper.
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.
A cost effective LISTEX™
application on Hot dogs
One unit treats 7.360
lbs
Cost-in-use is around
a penny per pound
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
Ripening period is the problem
Cross-contamination during
smearing
Low incidence - high numbers in the
final product
Mostly surface ripened cheeses
SMEARED CHEESE
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
Listeria occurance in a UK Salmon
plant
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
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.
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.
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
Growout
&
Transport
Live hang Immobili-
zation Bleed out
Scalding Picking Eviscerati
on
Immersio
n &
chilling
Sorting &
Aging
Processe
d
Products
Packing
Poultry Processing Steps
SALMONELEX™ Applications
• Chiller applications (pre-chiller)
• Trim prior to grinding
• Parts pre-packaging
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.
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
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