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Phagetherapy as alternative to antibiotictherapy in animal production

Joana AzeredoCentre of Biological EngineeringUniversity of Minho

www.ceb.uminho.pt/bbig

Symposium zum Europäischen Antibiotikatag 2014 (18th Nov 2014)

Presentation Outline

�Bacteriophages as biocontrol agents

�Advantages and disadvantages of phage therapy compared to antibiotherapy

�Examples: Phage therapy in poultry industry to control

� Salmonella

� Campylobacter

� E. coli

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Bacteriophages as biocontrol agents

� Bacteriophages (phage) are viruses that infect bacteria

� Phages are ubiquitous in the environment

� Phage can be highly specific or have a broad host range

� Phage specifically recognize and bind to their host through specific receptors

� Bacteriophages can be virulent (lytic) or temperate

T4- like bacteriophage

Only lytic phages have effective biocontrol properties

108 phage particles

Highlights in the development of phage as a potential therapeutic agent for bacterial infections (Levin and Bull, Nature, 2004)

Antibiotics surpassed phage

therapy

Renewed enthusiasm on account of

antibiotic resistance

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Bacteriophages as biocontrol agents

2005 2006 2007 2013

US Environmental Protection Agency approved AgriphageTM

FDA approved phage-based preparations for E. coli and Salmonella

control on RTE EcoShieldTM, SalmoFresh™

FDA approved

ListShieldTM

from Intralytix

and Listex TM

from MICREOS

FDA & USDA approve SALMONELEX™ as processing-aid against Salmonella

Discovery of bactriophages

d´Herelle and Twort

bacteriophages were for the

first time considered

generally recognized as safe

(GRAS)

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5

Bacteriophages as biocontrol agents

2005 2006 2007 2013

US Environmental Protection Agency approved AgriphageTM

FDA approved phage-based preparations for E. coli and Salmonella

control on RTE EcoShieldTM, SalmoFresh™

FDA approved

ListShieldTM

from Intralytix

and Listex TM

from MICREOS

FDA & USDA approve SALMONELEX™ as processing-aid against Salmonella

Approved phage products

Food additives

Poultry Productions

Human therapy

Bacteriophages as biocontrol agents

ADVANTAGES

OVER ANTIBIOTICSPHAGES

Highly specific

Do not disturb

normal flora

Ubiquitous

No

environmental

impact

Innocuous

Not toxic to

animals and

plants

Effective against

bacteria resistant

to antibiotics

Bind to bacterial

antigens/

(capsules, LPS)

Resistance strains

usually less virulent

Self replicating

Increase in

time

Lower doses

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Bacteriophages as biocontrol agents

DISADVANTAGES

OVER ANTIBIOTICSPHAGES

Highly specific

Do not disturb

normal flora

Ubiquitous

No

environmental

impact

Innocuous

Not toxic to

animals and

plants

Effective against

bacteria resistant

to antibiotics

Bind to bacterial

antigens/

(capsules, LPS)

Resistance strains

usually less virulent

Self replicating

Increase in

time

Lower doses

Bacteriophages as biocontrol agents

DISADVANTAGES

OVER ANTIBIOTICSPHAGES

Highly specific

Bind to bacterial

antigens/

(capsules, LPS)

Development of phage resistance Low efficacy

Low efficacy

Use of a cocktail

of different

phages

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Phage therapy in animal production: Poultry industry

High incidence of case reported outbreaks of Salmonella and Campylobacter

Salmonella

Campylobacter

E .coli

• High mortality rate

• Carcass rejection at slaughter

• Low feed conversion rate

• Weight loss

Represents very important economical losses in poultry industry

• High treatment cost

Avian Colibacillosis

N=214,286

N=91,034

Phage therapy in animal production: Poultry industry

Isolation of phages

Selection of phages

Phage characteriza

tion

Large scale phage

production

In vivo phage

efficacy trials

Isolation of phages

• Waste water

• Effluents

• Animal

• Poultry houses

Phages

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Phage therapy in animal production: Poultry industry

Isolation of phages

Selection of phages that

Phage characteriza

tion

Large scale phage

production

In vivo phage

efficacy trials

Selection of phages

A cocktail of phages with:

- Broad lytic spectra

- Complementary lytic spectra

- Cross resistances

- Lytic performance

Low High

Phage therapy in animal production: Poultry industry

Isolation of phages

Selection of phages

Phage characteriza

tion

Large scale phage

production

In vivo phage

efficacy trials

Phage characteriza

tion

RFLP analysis Phage genome sequencingTEM observation

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Phage therapy in animal production: Poultry industry

Isolation of phages

Selection of phages

Phage characteriza

tion

Large scale phage

production

In vivo phage

efficacy trials

Large scale phage

production

Tissue culture plates

2-5 L fermenters

In vivo phage

efficacy trials

Confined rooms Farms

Phage therapy in animal production: Campylobacter Control

Genome sequence:

Unique genes

Similarity to T4-like phages

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Phage therapy in animal production: Campylobacter Control

2

3

4

5

6

7

0 1 2 3 4 5 6 7

Nu

mb

er

of

C.c

oli

(lo

g 1

0 c

fu/g

)

Days post treatment

negative control

phage - oral gavage

phage - feed

Numbers of C.jejuni 2140CD1 in faeces from broilers orally

administered a phage cocktail by gavage (Carvalho et al., 2010)

Results of animal trials

Average 2-log reduction in Campylobacter

caecal content after 2 days of treatment

single dose by oral gavage

single dose incorporated in food

Phage cocktail incorporated in the birds’

feed led to an earlier reduction in

Campylobacter titer (2 days post-phage

administration)

Reduction of approximately 2 log cfu/g is

considered, by mathematical models to lead

to a 30-fold reduction in the incidence of

campylobacteriosis associated with

consumption of chicken meals.

In vivo acquisition of phage resistance

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Chickens groupCampylobacter titre

(log10cfu/g)

Resistant

strains

(%)

Non treated group 6.50 6

Treated group 4.29 13

Bacteria can naturally acquire phage resistance

�The resistant phenotype was not hindering the

ability of the Campylobacter to colonize the

chickens

�The majority of phage resistant strains reverted

to sensitive phenotypes

Phage therapy in animal production: Campylobacter Control

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Phage therapy in animal production: Salmonella Control

Salmonella Tiphimurium NCTC 12416 - subsp. I +

Salmonella NCTC 13349 - subsp. I +

Salmonella spp. SGSC 3047 - subsp. II +

Salmonella spp. SGSC 3039 - subsp. II +

Salmonella Arizonae 3063 - IIIa +/-

Salmonella Arizonae 83 - IIIa -

Salmonella spp. SGSC 3069 - subsp. IIIb +

Salmonella spp. SGSC 3068 - subsp. IIIb +

Salmonella spp. SGSC 3086 - subsp. IV +/-

Salmonella spp. SGSC 3074 - subsp. IV +

Salmonella Bongori SGSC 3103 - subsp. V +

Salmonella Bongori SGSC 3100 - subsp. V +

Salmonella spp. SGSC 3118 - subsp. VI +

Salmonella spp. SGSC 3116 - subsp. VI +

Salmonella spp. SGSC 3121 - subsp. VII +

Salmonella spp. SGSC 3120 - subsp. VII +

Escherichia coli n9 +/-

Escherichia coli n5 +/-

Escherichia coli CECT 434 (ATCC 25922) +/-

Escherichia coli BL21 +

Escherichia coli K12 +

Produce the phage in non-

pathogenic E. coli

Phage therapy in animal production: Salmonella Control

Salmonella caecal content (CFU/g) after 1 and 2 days post

administration of PVP-SE1 by oral gavage (PhageVet-P

report)

3 log reduction of

the treated group

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Phage therapy in animal production: APEC Control

Relative comparison (%) of the isolated strains according to susceptibility, intermediate susceptibility or resistance to a range ofantibiotics commonly used for therapy in poultry industry. AMP -Ampicillin; DO - Doxycycline; ENR - Enrofloxacin, STX -Sulphamethoxazole / Trimethropim, NA - Nalidixic acid, PIP - Pipemidicacid, TE - Tetracycline, OA - Oxolinic acid; AML- AmoxycillinOlveira et al., 2010

920 APEC isolated from 2003-2010 in Portugal are

highly resistant to antibiotics, over 80% are

resistant to tetracycline

Phage therapy in animal production: Avian Pathogenic Control

(A)phi F78E (Myoviridae) (B) phi F258E (Siphoviridae) (C) phi

F61E (Myoviridae)

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Phage therapy in animal production: Avian Pathogenic Control

Phage administration resulted in a decrease,

in average, of 25.0 % on chickens’ mortality

and of 41.7 % on morbidity

Phage administration resulted in a decrease,

in average, of 25.0 % on chickens’ mortality

and of 41.7 % on morbidityScore of injuries, mortality and morbidity in the treated and untreated group (Oliveira et al., 2010)

0

20

40

60

80

100

Morbidity Mortality

%

0

1

2

3

4

5

Pathology score

Scor

e

phage treated group

untreated group

Phage therapy in animal production: Avian Pathogenic Control

LARGE SCALE ANIMAL EXPERIMENTS5x107PFU/ml, administered in a single application, orally and by spray. In 11 flocks

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Conclusions

� Phages offer a great potential in controlling

bacterial pathogens in live animals

Facts and Challenges

Facts Challenges

Conclusions

� Phages offer a great potential in controlling

bacterial pathogens in live animals

Facts and Challenges

Facts Challenges

� Most phages are sensitive to low pH Neutralization of the pH

Phage encapsulation

� Phage resistant phenotypes

Phage application prior to slaughter

Environmental decontamination

� Phages are highly specific so the phage

cocktail should be designed are updated for

each particular application

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Conclusions Facts and Challenges

� Phages are highly specific so the phage

cocktail should be designed are updated for

each particular application

Animal Production Facilities

• Isolation of the pathogenic bacteria

Laboratory

• Selection of the most effective phage cocktail

Animal production

• Phage administration

Phage Library of well

characterized phages

Conclusions

� Phages offer a great potential in controlling

bacterial pathogens in live animals

Facts and Challenges

Facts Challenges

� Phages are sensitive to low pH Neutralization of the pH

Phage encapsulation

� Phage resistant phenotypes

Phage application prior to slaughter

Environmental decontamination

� Phages are highly specific so the phage

cocktail should be designed are updated for

each particular application What would be the regulatory frame for

this type of product?

Are pharma companies interested in

investing ?

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Acknowledgments

THANK YOU!

Ana Oliveira

Carla Carvalho

Sílvio Santos

Sanna Sillankorva

•Rob Lavigne (KULeuven)

•Ben Ganon (University of Bristol)

•Ian Sutherland (Edinburgh University)

•Victor Krylov (State Institute for Genetics of Industrial

Microorganisms)

•Hans Ackermann (Laval University)

•Andrew Kropinski (University of Guelph)

External collaborators