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

Questions & Follow-up

www.micreosfoodsafety.com

d.demeester@micreos.com