phage therapy: myth or reality ?escmid online lecture

22nd ECCMID - London 2012

Laurent DEBARBIEUX

Monday 2, April, 2012

Department of Microbiology

Phage Therapy: Myth or Reality ?

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Birth certificate of Phage Therapy

Date: 1920’s

Father: Félix d’Herelle (1873-1949)

Location: Institut Pasteur, Paris, France

Particular signs: first specific antibacterial treatment

1920’s-1940’s: world expansion (Brazil, Egypt, Georgia,…)

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1920’s-1940’s:

Doubts about the viral nature of bacteriophages

(“it is not a virus, it is an enzyme !”)

Lack of knowledge on how to manipulate bacteriophages

Specificity of bacteriophages not fully understood

Post-1940’s:

No conclusive data on reported treatments

Launch of the industrial production of antibiotics

Invention of electron microscope: Bacteriophages = Virus

“Phage Group” seeded in USA… birth of Molecular Biology

Decline of Phage Therapy

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Phage therapy was not totally abondonned

In the former Soviet Union, Georgia continued to develop Phage Therapy

… and today they are still treating regularly people with bacteriophages !

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1940’s-1980’s: in Eastern Europe countries

Industrial scale production of bacteriophages

Large range of clinical data (not in English)

Success rates in human treatments over 80%

Post-1980’s:

Collapse of the Soviet Union

Production sites mostly abandoned

Worldwide rise of antibiotic-resistant bacteria

Re-discovery of Phage Therapy (in UK first)

Meanwhile the knowledge on bacteriophages had increased

Phage Therapy, from 1940’s to date

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Family of Viruses of Microbes

96% of bacteriophages described are from Caudovirales

The T4 phage model

fibers

tail

capside

DNA

The most abundant biological entities on Earth (1031)

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Infectious cycle of bacteriophages

Phage proteins asantibacterials

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Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phagesFaruque et al., 2005, PNAS, vol 102, p1702

Bacteria/Bacteriophage arms race in environment

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21st century context

Antibiotics resistant bacteria are becoming a major public health

problem for which solutions are needed.

Davies, Microb Mol Biol Rev 2010, 74, 417-433

OldPhage

Therapy

NewPhage

Therapy ?

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Recent advances in Phage Therapy

In the agri-business:

Treatment of bacterial infections (plants)

Reduction of contaminations (meat products)

2006:

FDA (Food and Drug Administration) approval for a cocktail

anti-Listeria (meat products for humans)

2008:

extension to other food products (cheese)

2009:

publication of the first Phase II trial in humans (otitis)

2010:

Start of Phase II trial for diarrhea (Nestlé, Bangladesh)

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Pseudomonas aeruginosa and lungs infection

Opportunistic pathogen

Clinical isolates resistant to several antibiotics.

Both acute and chronic infections

Cystic fibrosis patients are often infected by P. aeruginosa.

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Escherichia coli and gut infections

E. coli: Commensal or Pathogen

Antibiotic resistance is continuously increasing

Specific targeting of pathogens with phages is appealing

(low impact on microbiota is expected)

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Phage Therapy on lungs infections

1) Bacteriophages

Isolation performed from wastewater (Paris area)

2) Animal model

acute lung infection caused by

P. aeruginosa (PAK strain)

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1) Amplification of bacteriophages infecting one given bacterial strain

2) Isolate individual plaques (at least three times)

water from Corbeilwater from Clichy water from Lagny

Bacteriophages characterization

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Bacteriophage / Bacteria interactions in broth

MOI test in broth

0,01

0,1

1

10

0 50 100 150 200 250 300time (min)

OD

at 6

00nm

NO

1/10-1

1/10-2

1/10-3

1/10-4

1/10-5

1/10-6

1/10-7

1/10-8

0.01

0.1

1

0 30 60 90 120 150 180 210 240 270 300

time (min)O

D (6

00nm

)

PAK-P1

PAK-P2

PAK-P3

1 108 bacteria + 1 phage=phage wins ! Comparison of kinetics of lysis


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L A B C D E

Bacteriophages: molecular characterization

RFLP analysis

kDa

116

66

45

35

25

18

14

Mass Spectrometry

(454 technology)

2 new groups

PAK_P1

PAK_P2

PAK_P3

PAK_P4

PAK_P5

Genome sequence

MW phage

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PAK strain

O:6

ND-2-4-6

ND-2-4-6

clinical isolates (serotypes)

O:4 O:9 O:10 O:11

Phage characterization

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Infection by 1.0x107 bacteria and 2H later different doses of phages

Bacteriophage treatment of lung infection in mice

PAK_P1

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time

Enlightening the Phage Therapy

Bioluminescent bacteria allow to follow-up the kinetics of

infection and treatment.

Advantages of bioluminescence:monitoring individual animals (no euthanasia)

quantification of the infection

PAK

timetime

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Infection at time 0by 1.0x107 bacteria

Infection at time 0by 1.0x107 bacteria

At time 2H

treatment with

1.0x108 bacteriophages

low

high

2H

2H

4H

4H6H

6H8H

8H

Debarbieux, J. Infect. Dis.2010, 201: 1096-1104

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What about a clinical strain (CHA) ?

PAK bacteriophage adapted to CHA strain by multiple passages

P3_CHA

Bacteriophage treatment is dose-dependantMorello, PLoS one 2011, 6: e16963

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Lungs status 20H post-infection

Bacteria Bacteriophages

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inflammatory markers

IL-6 KC

Lungs status 20H post-infection

Bacteriophage treatment reduces inflammatory response

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Histology of phage-treated lungs

bacteria

bacteria + phage

no bacteria

(PMNs, Lymphocytes, Infiltration

Alveolitis, Brochitis, Necrosis)

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antibodies directed against CHA strain (Ina Attree-Delic)

Immuno-histochemistry of phage-treated lungs

Not Infected

Infected

Phage treated

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Can bacteriophages prevent a lung infection ?

decay of bacteriophages

in the lungs 4 days pre-treatment

ensures full protection

Morello, PLoS one 2011, 6: e16963

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4 days preventive treatment: lungs status 20H post-infection

Bacteria Bacteriophages

IL-6 KC

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bacteria

4d phage + bacteria

no bacteria

Lungs histology of 4 days preventive treatments

(PMNs, Lymphocytes, Infiltration

Alveolitis, Brochitis, Necrosis)

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Not Infected

Infected

4 days phage pre-treated

Immuno-histochemistry of phage pre-treated lungs

Morello, PLoS one 2011, 6: e16963

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Bacteriophage adaptation

4/10

8/10

3/10

4/10

PAK_P3 on CHA strain (10%) P3_CHA (100%)

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PAK_P3 (vB_PaeM_PAK_P3) : 88097 bp

P3_CHA (vB_PaeM_P3_CHA) : 88097 bp (2 mutations)

Bacteriophage genome characterization

Morphogically related to FelixO1 phage, but genetically distant

2 new groups of virulents bacteriophages of P. aeruginosaPAK_P1, PAK_P2, PAK_P4, JG004PAK_P3, P3_CHA, PAK_P5, Kpp10

ORF 151: putative protein of 109 aa; no homologsORF 13: putative structural protein 440 aa–DUF3383/pfam11863;

(tail sheath / fiber protein)

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PAK_P3 vs P3_CHA (2 nucleotides difference)

Strong correlation between in vitro and in vivo infectivity

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Comparative study of bacteriophages

in silico vs in vitro vs in vivo correlations ?

In silico

Genome annotation and mining

In vitro In vivoEfficacy

of plaquing

Experimentationon murine model

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Phage representative EOP PAK strain

PAK_P1 1

PAK_P2 1

PAK_P4 1

PAK_P3 1

PAK_P5 1

LUZ19 0.23*ɸKZ >1*

LBL3 0.79*

* from R. Lavigne’s lab

Selection of bacteriophages infecting the PAK strain

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Family Genus Phage name Genome size Host strain

Podoviridae PhiKMV-like viruses LUZ19 44Kb PAO1

Myoviridae

KZ-like viruses ɸKZ 280Kb PAO1

PB1-like viruses LBL3 64Kb Aa245

Felix O1-like

PAK_P1 93 Kb PAK

PAK_P2 91 Kb PAK

PAK_P4 93Kb PAK

PAK_P3 88 Kb PAK

PAK_P5 85Kb PAK

Genomic comparison of bacteriophages

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High correlation between in vitro and in vivo data

All 8 bacteriophages but one (PhiKZ) behave in vivo

as expected from in vitro data.

Pulmonary Phage Therapy is not a myth

However, several questions remains:

Efficacy for chronic infections ?

Immune response if repeated treatments ?

Cystic fibrosis context ?

Conclusions from data obtained with the murine model

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CLB_P1 (40kb) 93% of homology with K1F (T7-like) on 85% of sequence

CLB_P2 (172kb) 95% of homology with JS98 (T4-superfamily) on 85% of sequence

CLB_P3 (50kb) 95% of homology with T1 (T1-like) on 85% of sequence

Enteroagregative Escherichia coli O104:H4 (55989)

What about gut infections ?

Isolation of 3 different bacteriophagesCLB_ P1 P2 P3

Mass Spec. of MCP

Genomes sequence

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Bacteriophages Activity in vitro on 55989

Rapid lysis followed by growth of resistant bacteria (CLB_P1 and CLB_ P3)

No phage-resistant bacteria with CLB_P2 and the cocktail

Bacteriophages are also infectious in anaerobic conditions

0 1 2 3 4 5 6 7 8 9 10 110.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

time (h)

OD

600n

m

CLB-P1 CLB-P2 CLB-P3 cocktail

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Bacteriophages infect in vitro biofilms

Cumulative effect of the cocktail of the 3 bacteriophages

- +

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Bacteriophages infect in vivo biofilms

Without bacteriophages With bacteriophages

Maura, Environmental Microbiology, Nov 28, 2011

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In feces bacteria are unaffected

d-5 d-3 d0 d1 d18

E. coli cocktail

streptomycin

Active replication of bacteriophages in vivo

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 181.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

time (days post phage administration)[E

.col

i 559

89 S

tr N

al] (

cfu/

g of

faec

es)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 181.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

time (days post phage administration)

[pha

ges]

(pfu

/g o

f fae

ces)

Mice not colonized Mice colonized Mice colonized Mice colonized+ phages

Bacteriophages impact on the gut

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Upper small intestin

Lower small intestin

Upper large intestin

Lower large intestin

Do feces reflect the whole intestine ?

In depth analysis of bacterial colonization

Kinetics studies on bacteria and bacteriophages

? ? ? ?

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The 55989 strain is colonizing both small and

large intestines without damaging epithelium

Kinetics of colonization

Wit

ho

ut

55

98

9W

ith

55

98

9

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Impact of bacteriophages on ileal colonization

Dose-dependant reduction… but temporary

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Dose-dependant reduction and temporary effects both confirmed

Impact of bacteriophages on ileal colonization (qPCR)

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Parameters affecting bacteriophages efficacy in the gut

1) phage resistant bacteria ?No, because of we used a cocktail

2) mouse microbiota ?No, same results from axenic mice monocolonized

3) biofilms ?Unlikely (in vitro and in vivo biofilms)

4) inhibitors of phages in the gut ?No, ex-vivo experiments (gut sections)

5) intracellular stage of 55989Unlikely (not seen in immunohistochemistry)

6) permissivity of the host along the gut is not uniform ?YES !

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Permissivity to 55989 is not uniform along the gut

CLB_P1 and CLB_P3 << CLB_P2 in stationary phase vs exponential

CLB_P1 and CLB_P3 << CLB_P2 in feces versus ileal samples

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With a gut colonization model we demonstrated that:

Bacteriophages can replicate in vivo

Host “state” is affecting bacteriophages replication in vivo

No direct correlation between in vitro and in vivo data

Conclusions from data on the intestinal model

Intestinal Phage Therapy is not a myth

However, several questions remains:

How to predict in vivo bacteriophage efficacy ?

Immune response if repeated treatments ?

Roles of inflammatory/diarrheal context ?

No myth anymore… even if the picture in not entirely clear,

Phage Therapy is making its way back to medicine

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IBBA project: People and Funding

Collaborators:

Lhousseine TOUQUI (I. Pasteur)

Chantal Le BOUGUENEC (I. Pasteur)

Michel HUERRE (I. Curie)

Isabelle SERMET (H. Necker)

Aleksander EDELMAN (H. Necker)

Rob LAVIGNE (K.U. Leuven)

Current Lab members Former Lab members

Matthieu GALTIER Damien MAURA

Marine HENRY Eric MORELLO

Emilie SAUSSEREAU

www.pasteur.fr/research/phages

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phage therapy: myth or reality ?escmid online lecture

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