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22nd ECCMID - London 2012
Laurent DEBARBIEUX
Monday 2, April, 2012
Department of Microbiology
Phage Therapy: Myth or Reality ?
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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
Infectious cycle of bacteriophages
Phage proteins asantibacterials
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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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
Bacteriophages characterization
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Phage Therapy: Myth or Reality ?
Bacteriophages characterization
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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
Lungs status 20H post-infection
Bacteria Bacteriophages
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Phage Therapy: Myth or Reality ?
inflammatory markers
IL-6 KC
Lungs status 20H post-infection
Bacteriophage treatment reduces inflammatory response
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Phage Therapy: Myth or Reality ?
Histology of phage-treated lungs
bacteria
bacteria + phage
no bacteria
(PMNs, Lymphocytes, Infiltration
Alveolitis, Brochitis, Necrosis)
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Phage Therapy: Myth or Reality ?
antibodies directed against CHA strain (Ina Attree-Delic)
Immuno-histochemistry of phage-treated lungs
Not Infected
Infected
Phage treated
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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
4 days preventive treatment: lungs status 20H post-infection
Bacteria Bacteriophages
IL-6 KC
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Phage Therapy: Myth or Reality ?
bacteria
4d phage + bacteria
no bacteria
Lungs histology of 4 days preventive treatments
(PMNs, Lymphocytes, Infiltration
Alveolitis, Brochitis, Necrosis)
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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
Bacteriophage adaptation
4/10
8/10
3/10
4/10
PAK_P3 on CHA strain (10%) P3_CHA (100%)
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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
PAK_P3 vs P3_CHA (2 nucleotides difference)
Strong correlation between in vitro and in vivo infectivity
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Phage Therapy: Myth or Reality ?
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 Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Slide withheld at request of author
Online Lecture Library
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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
Bacteriophages infect in vitro biofilms
Cumulative effect of the cocktail of the 3 bacteriophages
- +
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Phage Therapy: Myth or Reality ?
Bacteriophages infect in vivo biofilms
Without bacteriophages With bacteriophages
Maura, Environmental Microbiology, Nov 28, 2011
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Phage Therapy: Myth or Reality ?
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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
Impact of bacteriophages on ileal colonization
Dose-dependant reduction… but temporary
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Phage Therapy: Myth or Reality ?
Dose-dependant reduction and temporary effects both confirmed
Impact of bacteriophages on ileal colonization (qPCR)
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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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Phage Therapy: Myth or Reality ?
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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