Phage Therapy of Pseudomonas infections
The
present invention relates to novel bacteriophages, compositions
comprising the same, their manufacture, and the uses thereof.
The
invention is particularly adapted for the treatment of an
infection in a
mammal and for improving a subject condition by modifying the flora in
said subject.
BACKGROUND OF THE INVENTION
Bacteriophages (or phages) are small viruses displaying the ability to
infect and kill bacteria while they do not affect cells from other
organisms. Initially described almost a century ago by William Twort,
and independently discovered shortly thereafter by Felix d'Herelle, more
than 6000 different bacteriophages have been discovered so far and
described morphologically, including bacterial and archeal viruses. The
vast majority of these viruses are tailed while a small proportion are
polyhedral, filamentous or pleomorphic. They may be classified according
to their morphology, their genetic content (DNA vs. RNA), their
specific host, the place where they live (marine virus vs. other
habitats), and their life cycle. As intra-cellular parasites of
bacterial cells, phages display different life cycles within the
bacterial host: lytic, lysogenic, pseudo-lysogenic, and chronic
infection (Weinbauer, 2004; Drulis-Kawa, 2012). Lytic phages cause lysis
of the host bacterial cell as a normal part of their life cycles.
Lysogenic phages (also termed temperate phages) can either replicate by
means of the lytic life cycle and cause lysis of the host bacterium, or
they can incorporate their DNA into the host bacterial DNA and become
noninfectious prophages. Whatever the type of cycle of a phage life, the
first step is the attachment to receptors of the bacterial cell wall
before phage material may enter the bacteria. This specific process
influences the spectrum of the possible phage-bacteria interactions.
Bacteriophages are commonly used as research tools to modify bacteria in laboratory experiments.
Because
of their target host cell specificity, the use of phages as a therapy
to treat acute and chronic infections has been considered, particularly
in dermatology,
ophthalmology, urology, stomatology, pediatrics,
otolaryngology or surgery. This concept of therapeutic use of phages to
treat bacterial infection was, however, highly controversial from the
very beginning and not widely accepted by the public or medical
community. Early studies were widely criticized for lack of appropriate
controls and inconsistent results. The lack of reproducibility and many
conflicting results obtained in the various published studies led the
Council on Pharmacy and Chemistry of the American Medical Association to
conclude that the evidence for the therapeutic value of lytic filtrates
was for the most part contradictory, unconvincing, and recommended
additional research to confirm its purported benefits.
Since
the introduction of antibiotics in the 1940s, little attention was paid
to this field of therapeutics, especially in the Western world. But the
extensive use of antibiotics has led to the widespread emergence and
spread of antibiotic-resistant bacteria around the world, causing
increasingly serious problems. It has therefore become a major
therapeutic challenge to overcome the limited therapeutic options
remaining to treat major multi-drug resistant microbes.
In
addition, many pathogenic microorganisms reside within bio films, which
bio films cause additional problems when designing new anti-microbial
agents. In this regard, bacteria growing as a biofilm rather than in
single-celled ("planktonic") forms tend to be particularly resistant to
anti-microbial agents and to be particularly difficult for the host
immune system to render an appropriate response.
Since
its initial discovery in the late 19th century (Fordos 1859), the
Gram-negative bacterium Pseudomonas aeruginosa has gained a notorious
place in the list of infamous human pathogens (Williams and al, 1894,
Freeman and al, 1916). The arrival of the antibiotic era largely
palliated the previously fatal outcome of acute infections in healthy
patients. Only a relative improvement has been achieved in the
eradication of chronic infections, which develop mainly in individuals
suffering from cystic fibrosis or severe burns or who are
immunocompromised (Gang et al, 1999, Jones and al, 2010). Two
intrinsically related factors in the fatal outcome of infection in these
patients are the rapid prescription of inappropriate antibiotic
treatments and the development or acquisition of multidrug-resistant
strains. While the use of (an) appropriate antibiotic(s) has been
reported as an essential factor in the eradication of P. aeruginosa
infections (Kang and al, 2005, Micek and al, 2005), conversely,
antibiotic abuse significantly contributes to increasing resistance by
exerting a continuous selective pressure for the acquisition of such
capabilities. Antibiotics alone do not account for the high prevalence
of multidrug-resistant variants: P. aeruginosa has multiple,
chromosomally encoded intrinsic mechanisms of resistance, including low
permeability of the cell envelope and numerous multidrug efflux pumps.
Another major factor accounting for the successful invasive behavior and
persistence of this bacterium is its high adaptability, allowing rapid
colonization of different environments.
Furthermore,
pathogenic bacteria such as P. aeruginosa are able to form biofilms,
which contribute to their increased resistance to antibiotics. Such
biofilms may comprise more than one type of bacteria supported and
surrounded by an excreted extracellular matrix, and assist bacteria to
colonize various surfaces. Biofilms allow bacteria to attach to surfaces
and to reach population densities which would otherwise be
unsupportable, imparting increased resistance to not only antibiotics
but many environmental stresses including toxins such as heavy metals,
bleaches and other cleaning agents. It is known that bacteria within
biofilms can be 100 to 1000 times more resistant to antibiotics than the
same strain of bacteria growing in planktonic forms. Such an increased
resistance means that bacteria that are apparently sensitive to
antibiotics in a laboratory test may be resistant to therapy in a
clinical setting. Even if some are cleared, biofilms may provide
resistant reservoirs permitting rapid colonization once antibiotics are
no longer present. It is therefore obvious that biofilms are major
factors in many human diseases. Chemical treatments are unsuited to use
against biofilms since this is precisely what they have evolved to
counter. Physical abrasion does provide a mean to disrupt biofilms.
Unfortunately, many surfaces where biofilms supports bacterial
pathogenesis are poorly suited to rigorous abrasion, i.e. bones, joints,
implanted medical devices, etc. For example, the surfaces of wounds or
burns are extremely sensitive and delicate. Even where abrasion is both
suitable and in routine use, clearing of biofilms is limited. Oral
plaque on the surface of teeth is a biofilm and is partially cleared by
regular brushing. However, bacteria are maintained on unbrushed surfaces
(for example in the gaps between teeth) and can reco Ionize
cleared
surfaces both rapidly and effectively. From this, it is clear that
existing approaches to clearing bio films are of limited efficacy.
The
capability for quick adaptation and their ability to form bio films are
the main reasons that identify P. aeruginosa as opportunistic
pathogens. They have acquired the status of hospital pathogens, and may
be isolated from clinical samples taken from the wounds, sputum,
bladder, urethra, vagina, ears, eyes and respiratory tract. The
emergence of resistance to the most powerful new antibiotics in such
clinical P. aeruginosa strains, occurring even during treatment, makes
the fight with P. aeruginosa hospital pathogens a great problem.
Furthermore,
it has been reported that the pathological or physiological condition
of a subject is influenced by the balance of microorganisms in the flora
of the subject. Accordingly, modifying the microbial flora, or
modifying said balance, or restoring said balance, by destroying P.
aeruginosa population, is also a valuable approach for improving a
subject condition.
Therefore, there is a great need for
new antibacterial agents or compositions that can be used to destroy P.
aeruginosa strains, even when organized in bacterial biofilms, suitable
for use in human or animal therapy, as well, as for decontaminating
materials.
SUMMARY OF THE INVENTION ( invention ????)
The
inventors have isolated and characterized new bacteriophages presenting
specific lytic activity to Pseudomonas aeruginosa (P. aeruginosa), and
which can be used as active agents in pharmaceutical or veterinary
preparations, particularly to treat P. aeruginosa bacterial infections
or to modify microbial balance in a subject. The new bacteriophages of
the invention exhibit strong lytic activity, high selectivity, and can
by combined to induce controlled destruction of a very large spectrum of
P. aeruginosa cells.
An object of the invention is to
provide antibacterial compositions comprising at least one, preferably
at least two bacteriophages having lytic activity against a Pseudomonas
aeruginosa (P. aeruginosa) strain, said bacteriophages being selected
from the bacteriophages having a genome comprising a nucleotide sequence
of anyone of SEQ ID NOs: 1 to 13 or a sequence having at least 90%
identity thereto.
A further object of the invention relates to a
bacteriophage having lytic activity to a Pseudomonas aeruginosa (P.
aeruginosa) strain and having a genome comprising a nucleotide sequence
selected from anyone of SEQ ID NOs: 1 to 13 or a sequence having at
least 97% identity thereto.
The bacteriophages of the invention
exhibit lytic activity to multi drug resistant strains of P. aeruginosa,
in particular to antibiotic resistant pathogenic strains, such as
cephalosporinase, carbenicillinases and extended-spectrum /?-lactamases
(Strateva T. and Yordanov D. 2009).
In another aspect, the
invention is related to a bacteriophage having lytic activity to a
pathogenic P. aeruginosa strain, wherein the bacteriophage is specific
for P. aeruginosa, active against antibiotic-resistant P. aeruginosa
strains, and has a productive lytic effect below 20.
The
invention further concerns an isolated nucleic acid contained in a
bacteriophage of the invention, preferably an isolated nucleic acid
molecule comprising a nucleotide sequence selected from anyone of SEQ ID
NOs: 1 to 13 or a sequence having at least 97%) identity thereto, as
well as an isolated polypeptide encoded by said nucleic acid.
Another object of the invention is a composition comprising a nucleic acid or polypeptide as defined above.
The
compositions of the invention typically further comprise a
pharmaceutically or veterinary acceptable excipient or carrier. They may
be liquid, semi-liquid, solid or lyophilized.
Another
object of the invention relates to a bacteriophage, nucleic acid,
polypeptide or composition as defined above, for use in the treatment of
an infection in a mammal, for modifying the microbial flora in a
mammal, for decontaminating a material and/or for killing a P.
aeruginosa bacterium or for compromising the integrity of a bacterial
bio film.
The invention relates also to the use of one or
several lytic bacteriophages to improve a subject condition by modifying
the microbial flora in said subject. The microbial flora may be
modified by correcting, adapting or restoring a proper balance of
microorganisms in said flora.
The invention also relates to a
method for treating an infection in a mammal, comprising the
administration to said mammal of at least one bacteriophage, nucleic
acid, polypeptide or composition as defined above.
The
invention also relates to a method for treating a surface or material
suspected of being contaminated with a P. aeruginosa bacterium,
comprising applying to said surface or material at least one
bacteriophage, nucleic acid, polypeptide or composition as defined
above. The surface or material may be a surface of any device, vessel or
laboratory material, cloth, etc.
A further object of the
invention relates to a kit comprising a composition as defined above and
a means for applying the same to a subject or surface.
Another
object of the invention relates to a method for predicting or
determining efficacy of a bacteriophage therapy in a subject, wherein
the method comprises determining in vitro a lytic activity of one or
more bacteriophages of the invention to a P. aeruginosa strain from a
sample of said subject, a lytic activity of one or more bacteriophages
of the invention to at least one P. aeruginosa strain from said sample
being indicative of an efficient treatment. The method further
optionally comprises the step of treating the subject with at least one
bacteriophage having a lytic activity to a P. aeruginosa strain from a
sample of said subject.
In another aspect, the invention
provides a method for selecting a subject or determining whether a
subject is susceptible to benefit from a bacteriophage therapy, wherein
the
method comprises the step of determining in vitro a lytic
activity of one or more bacteriophages of the invention to a P.
aeruginosa strain from a sample of said subject, a lytic activity of one
or more of said bacteriophages to at least one P. aeruginosa strain
being indicative of a responder subject.
The invention
may be used in any mammal, preferably in human beings, or to treat any
material, including laboratory materials or medical devices.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: In vitro efficacy of Bacteriophages of the invention on various combinations of P. aeruginosa strains.
Figure 2: In vivo efficacy of Bacteriophages of the invention on various combinations of P. aeruginosa strains.
Figure 3: Efficacy of bacteriophages of the invention in vivo on Is580 P. aeruginosa strain-mediated infection.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to novel bacteriophages, components
thereof, compositions comprising the same, their manufacture, and the
uses thereof as antibacterial agents, particularly for the treatment of
an infection in a mammal and for improving a subject condition by
modifying the microbial flora in said subject.
Definitions
To facilitate understanding of the invention, a number of terms are defined below.
As
used herein, the term "bacteriophage" or "phage" refers to a functional
phage particle comprising a nucleic acid genome packaged in a
proteinaceous envelope or capsid. The term also refers to portions of
the bacteriophage, including, e.g., a head portion, or an assembly of
phage components, which provide substantially the same functional
activity.
The term "phenotypic characteristic" designates
more preferably the morphology and/or host-range of a bacteriophage.
Methods for phenotyping bacteriophages are well known per se in the part
and include, for example, determining bacterial host range and/or
activity against the biofilm produced by certain bacterial strains.
The
term "lytic activity" as used in the invention designates the property
of a bacteriophage to cause lysis of a bacterial cell. The lytic
activity of a bacteriophage can be tested on P. aeruginosa strains
according to techniques known per se in the art (see also experimental
section).
The term "variant" of a reference bacteriophage
designates bacteriophages having variation(s) in the genomic sequence
and/or polypeptide(s) encoded thereby as compared to said reference
bacteriophage, while retaining the same phenotypic characteristic as the
reference bacteriophage. Variants typically comprise e.g., silent
mutations, conservative mutations, minor deletions, and/or minor
replications of genetic material, and retain phenotypic characteristics
of the reference bacteriophage. In a preferred embodiment, the variant
of the invention retain any observable characteristic or property that
is dependent upon the genome of the bacteriophage of the invention, i.e.
phenotypic characteristics of said bacteriophage and/or lytic activity
against the P. aeruginosa strains. Preferred variants have less than 5%
nucleic acid variation as compared to the genome of the reference
bacteriophage, even more preferably less than 4%, more preferably less
than 2%. Alternatively, or in combination, variants have preferably less
than 5% amino acid variation in a coded polypeptide sequence as
compared to a polypeptide of the reference bacteriophage.
The
term "% identity" in relation to nucleic acid or amino acid sequences
designates the level of identity or homology between said sequences and
may be determined by techniques known per se in the art. Typically, the
%identity between two nucleic acid or amino acid sequences is determined
by means of computer programs such as GAP provided in the GCG program
package (Program Manual for the Wisconsin Package,
Version 8,
August 1996, Genetics Computer Group, 575 Science Drive, Madison,
Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (1970), Journal
of Molecular Biology, 48, 443-453). With settings adjusted to e.g., DNA
sequences (particularly: GAP creation penalty of 5. 0 and GAP extension
penalty of 0.3), nucleic acid molecules may be aligned to each other
using the Pileup alignment software available as part of the GCG program
package. The %identity between two sequences designates the identity
over the entire length of said sequences.
The term
"fragment" of a nucleic acid designates typically a fragment having at
least 10 consecutive nucleotides of said nucleic acid, more preferably
at least 15, 20, 25, 30, 35, 40, 50 or more consecutive nucleotides of
said nucleic acid.
The term "fragment" of a polypeptide
designates typically a fragment having at least 5 consecutive amino
acids of said polypeptide, more preferably at least 10, 15, 20, 30, 40,
50 or more consecutive amino acids of said polypeptide.
The terms "ESBL P. aeruginosa strain" refers to cephalosporinase and/or extended-spectrum/?-lactamases producing P. aeruginosa strains, including various forms
of antibiotic resistance such as AmpC /?-lactamase or Class A
carbenicillin hydro lyzing β-lactamases, etc.
The term
"specific" or "specificity" in relation to a bacteriophage refers to the
type of host that said bacteriophage is able to infect. Specificity is
usually mediated by the tail fibers of bacteriophages, that are involved
in the interaction with receptors expressed on cells. A bacteriophage
"specific" for P. aeruginosa more preferably designates a bacteriophage
which can infect one or several P. aeruginosa strains and which cannot
infect non- . aeruginosa bacteria under physiological conditions.
As
used herein, the term "polypeptide" refers to polypeptides of any size,
including small peptides of e.g., from 5 to 20 amino acids, longer
polypeptides, proteins or fragments thereof.
The term "PLE" or
"Productive Lytic Effect" designates the ratio between burst size and
productive lytic time of a given bacteriophage. Burst size and
productive lytic time are parameters defining phage-host interaction and
correspond, respectively, to the mean yield of bacteriophage particles
produced by infection of one bacterium by one phage, and to the time
taken by a free bacteriophage to lyse a bacterial cell.
In the context of the present specification, the term "isolated bacteriophage'''
should be considered to mean material removed from its original
environment in which it naturally occurs. In relation to a
bacteriophage, the term designates, particularly, a phage that is e.g.,
cultivated, purified and/or cultured separately from the environment in
which it is naturally located. In relation to a nucleic acid or
polypeptide, the term "isolated" designates e.g., a nucleic acid
molecule or polypeptide which is separated from at least some of the
components of its natural environment such as, e.g., a protein, lipid,
and/or nucleic acid.
The terms "pharmaceutically or
veterinary acceptable" as used herein refers to any material (e.g.,
carrier, excipient or vehicle) that is compatible for use in a mammalian
subject. Such includes physiologically acceptable solutions or vehicles
that are harmless or do not cause any significant specific or
non-specific immune reaction to an organism or do not abrogate the
biological activity of the active compound. For formulation of the
composition into a liquid preparation, saline, sterile water, Ringer's
solution, buffered physiological saline, albumin infusion solution,
dextrose solution, maltodextrin solution, glycerol, ethanol, and
mixtures thereof may be used as a pharmaceutically or veterinary
acceptable excipient or carrier. If necessary, other conventional
additives such as thickeners, diluents, buffers, preservatives, surface
active agents, antioxidants and bacteriostatic agents may be added.
Further, diluents, dispersants, surfactants, binders and lubricants may
be additionally added to the composition to prepare injectable
formulations such as aqueous solutions, suspensions, and emulsions, oral
formulations such as pills, capsules, granules, or tablets, or powdered
formulations.
As used herein, "PFU' means plaque forming
unit, as it is well defined in the art. Lytic bacteriophages lyse the
host cell, causing a zone of clearing (or plaque) on a culture plate.
Theoretically, each plaque is formed by one phage and the number of
plaques multiplied by the dilution factor is equal to the total number
of phages in a test preparation.
The term "treatment" or
"therapy" designates a curative or a prophylactic treatment of a
disease. A curative treatment is defined as a treatment that results in a
cure of a disease, or a treatment that alleviates, reduces, stabilizes,
or eliminates the symptoms of a disease or the suffering that it
causes, directly or indirectly, or that improves a subject condition or
reduces progression of a disease. A prophylactic treatment comprises
both a treatment resulting in the prevention of a disease and a
treatment reducing and/or delaying the incidence of a disease or the
risk of its occurrence.
The term "mammal" includes human
subjects as well as non-human mammals such as pets (e.g., dogs, cats),
horses, ruminants, sheeps, goats, pigs, etc.
The term
"biofilm" as used herein designates a heterogeneous bacterial formation
growing on various surfaces; preferably a bacterial community growing
embedded in an exopolysaccharide matrix adhered onto solid biological or
non-biological surfaces.
The term "compromise" as used herein
refers to any alteration of the integrity. By compromising a bacterial
biofilm, it is understood a penetration of the biofilm by bacteriophage,
an infection of bio film-associated bacteria and/or a lysis thereof
and/or a partial or an entire clearing of the biofilm (i.e., by stopping
colonization and/or disrupting bio films).
The term
"sample", as used herein, means any sample containing cells. Examples of
such samples include fluids such as blood, plasma, saliva, or urine as
well as biopsies, organs, tissues or cell samples. The sample may be
treated prior to its use.
As used herein, the term "subject" or
"patient" refers to an animal, preferably to a mammal, even more
preferably to a human, including adult and child. However, the term
"subject" also encompasses non-human animals, in particular mammals such
as dogs, cats, horses, cows, pigs, sheeps and non-human primates, among
others.
The term "efficacy" of treatment or "response" to a
bacteriophage therapy as used herein refers to a treatment which results
in a decrease in the number of P. aeruginosa strains in a subject after
bacteriophage treatment when compared to the number of P. aeruginosa
strains before treatment. A "good responder" subject refers to a subject
who shows or will show a clinically significant recovery when treated
with a bacteriophage therapy.
The term "Cocktail" or
composition of bacteriophages designates a combination of different
types of bacteriophages. The bacteriophages in a cocktail/composition
are preferably formulated together, i.e., in a same vessel or packaging,
although they may be used as kits of parts wherein the (or some of the)
bacteriophages are formulated or packaged separately and combined when
used or administered.
Description of embodiments
The
present invention is related to novel bacteriophage therapies. More
particularly, the present invention relates to novel bacteriophages
having a high specificity against Pseudomonas aeruginosa strains, their
manufacture, components thereof, compositions comprising the same and
the uses thereof in phage therapy.
Bacteriophages:
In a first aspect, the invention discloses the isolation and
characterization of novel bacteriophages that are specific for P.
aeruginosa strains and present, either alone or in combination(s),
remarkable host range spectrum of lytic activity. These bacteriophages
have been selected from environmental samples, isolated, sequenced, and
characterized. As indicated, the bacteriophages are, individually and in
combination(s), active against P. aeruginosa strains. They are
remarkable effective against pathogenic P. aeruginosa strains, such as
antibiotic-resistant P. aeruginosa strains, such as an ESBL P.
aeruginosa strain. Furthermore, bacteriophages of the invention have a
remarkable productive lytic effect ("PLE") below 20, more preferably
below 15 and still more preferably between 0.3 and 15. Moreover,
the bacteriophages of the invention are specific for P. aeruginosa
strains, i.e., they do not cause lysis of non- . aeruginosa bacteria. As
will be illustrated further, the invention shows that these
bacteriophages can be combined and formulated in conditions suitable for
use as pharmaceutical or veterinary agents to exhibit targeted and very
potent antibacterial effect against a controlled spectrum of P.
aeruginosa strains.
More specifically, the following
bacteriophages have been selected and characterized. Their corresponding
nucleic acid sequences are also indicated.
Table 1
The
lytic profile of these bacteriophages has been determined on a broad
number of P. aeruginosa strains. These bacteriophages have been selected
for their potency and combination potential, as disclosed in the
following table. In this table, the lytic effect of the bacteriophages
on reference and pathogen-resistant strains are presented, to confirm
the high lytic potential.
Table 2
Further
results on highly resistant strains from wound or burn are presented
below, further confirming the remarkable activity profile of the
bacteriophages of the invention, and their complementarity.
Table 3
*CAR : Class ATB Resistance
As
can be seen from tables 2 and 3, the phages have individually very
strong lytic power, and combinations (or cocktails) of these
bacteriophages may be produced that are able to kill all of the tested
P. aeruginosa strains, thereby producing broad spectrum antibacterial
compositions.
As an illustration, a cocktail of all 13 phages
of the invention is able to effectively kill all bacteria listed in
Table 2 and Table 3.
Moreover, the specificity of the
bacteriophages has been tested on many non-P. aeruginosa strains. More
particularly, the experimental section demonstrates that the
bacteriophages of the invention have no lytic effect on any bacteria
selected from Escherichia coli, Acinebacter baumanii, Enterobacter
aerogenes, Enterobacter asburiae, Enterobacter cloacae, Klebsiella
pneumonia, Porteus mirabilis, Staphylococus aureus, Stenotrophomonas
maltophila and/or Serratia marcescens.
A particular
object of the invention thus resides in a bacteriophage having lytic
activity to a P. aeruginosa strain and having a genome comprising a
nucleotide sequence selected from anyone of SEQ ID NOs: 1 to 13 or a
sequence having at least 97% identity thereto, preferably at least 98%
or 99% identity thereto.
The bacteriophages of the invention may
be cultured, expanded, isolated, purified, and used in e.g., phage
therapy of P. aeruginosa-mediated disorders, as will be disclosed in
more details below. Furthermore, variants of these bacteriophages
retaining a phenotypic (e.g., specificity and lytic activity) of the
bacteriophages can be produced and/or isolated by techniques known per
se in the art.
The bacteriophages of the invention can be
prepared by standard culture, isolation and purification methods. For
example, P. aeruginosa producing bacteria are cultured, infected by a
sample of a bacteriophage, and then treated to remove bacterial cells
and debris. The enriched bacteriophage solution can be plated in a
medium, for example agar medium, with embedded susceptible host strains
of P. aeruginosa to obtain plaques. Then, single plaque can be picked
out for subsequent bacteriophage purification and amplification. One or
more cycles of selective amplification of bacteriophages of the
invention may be performed, for example by mixing bacteriophages with
the competent P. aeruginosa, followed by addition of a growth medium and
incubation at selected test growing conditions. Following
centrifugation, the cleared amplified supernatant is filtered through
filter and subjected to another cycle of selective amplification or
tested for presence of lytic activity.
The titer of phage
in a suspension and the visualization of plaque morphology of
bacteriophages of the invention may then be assessed by known methods,
for example by plaque counting. Additionally, processing bacteriophages
of the invention in various forms (liquid, lyophilized, etc.) for
short-, long-, freeze- or any other kind of storage can be carried out
by any suitable method as it is well-known in the art (see e.g., Clark,
1962).
The activity of the bacteriophages of the
invention can be assessed by methods well-known in the art, such as
plaque assay also known as double agar method, based on the growing of
bacteriophage with potential host bacteria and followed by assessing
their ability to kill the host bacterial cell. In the plaque assay
method, the bacteriophage induces lysis of target P. aeruginosa strains
after a period of incubation in soft agar medium, resulting in zones of
clearing on the plate known as plaques.
In a particular
embodiment, the invention is related to BP 1384 bacteriophage, or any
variant thereof. BP 1384 bacteriophage, or any variant thereof, can be
produced or expanded in e.g., P. aeruginosa strain PAOl . BP1384, or any
variant thereof, is specific and has lytic activity against LMG24882,
LMG24883, LMG24886, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898
and/or LMG24909 strains. BP 1384 comprises a genome comprising a
sequence as set forth in SEQ ID NO: 1 or having at least 80% identity,
more preferably at least 85% identity, and still more preferably
90%>, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1.
It is also provided an isolated nucleic acid sequence from BP 1384
bacteriophage, or variant thereof. The invention also encompasses
isolated polypeptides encoded by BP 1384 bacteriophage, or variant
thereof, or encoded by an isolated nucleic acid sequence from BP 1384
bacteriophage of the invention. BP 1384 bacteriophage of the invention
is also characterized by a PLE below 20, more preferably below 15 and
still more preferably of around 6.2.
In another
particular embodiment, the invention is related to BP 1429
bacteriophage, or any variant thereof. BP 1429 bacteriophage, or any
variant thereof, can be produced or expanded in e.g., P. aeruginosa
strain PAOl . BP1429, or any variant thereof, is specific and has lytic
activity against LMG24882, LMG24891, LMG24892, LMG24893, LMG24896,
LMG24898 and/or LMG24916 strains. BP1429 comprises a genome comprising a
sequence as set forth in SEQ ID NO: 2 or having at least 80%>
identity, more preferably at least 85% identity, and still more
preferably 90%>, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO: 2. It is also provided an isolated nucleic acid sequence from BP
1429 bacteriophage, or variant thereof. The invention also encompasses
isolated polypeptides encoded by BP 1429 bacteriophage, or variant
thereof, or encoded by an isolated nucleic acid sequence from BP 1429
bacteriophage of the invention. BP 1429 bacteriophage of the invention
is also characterized by a PLE below 20, more preferably below 15 and
still more preferably of around 0,70.
In still another
aspect, the invention is related to BP 1430 bacteriophage, or any
variant thereof. BP1430 bacteriophage, or any variant thereof, can be
produced or expanded in e.g., P. aeruginosa strain PAOl . BP1430, or any
variant thereof, is specific and has lytic activity against LMG24882,
LMG24883, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898, LMG24901
and/or LMG24918 strains. BP1430 comprises a genome comprising a sequence
as set forth in SEQ ID NO: 3 or having at least 80% identity, more
preferably at least 85% identity, and still more preferably 90%>,
92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3. It is also
provided an isolated nucleic acid sequence from B1430 bacteriophage, or
variant thereof. The invention also encompasses isolated polypeptides
encoded by BP 1430 bacteriophage, or variant thereof, or encoded by an
isolated nucleic acid sequence from BP 1430 bacteriophage of the
invention. BP 1430 bacteriophage of the invention is also characterized
by a PLE below 20, more preferably below 15 and still more preferably of
around 3.
In another aspect, the invention is related to
BP 1433 bacteriophage, or any variant thereof. BP1433 bacteriophage, or
any variant thereof, can be produced or expanded in e.g., P. aeruginosa
strain PAOl . BP1433, or any variant thereof, is specific and has lytic
activity against LMG24882, LMG24883, LMG24886, LMG24887, LMG24891,
LMG24892, LMG24893, LMG24896, LMG24896, LMG24905, LMG24909 and/or
LMG24916 strains. BP 1433 comprises a genome comprising a sequence as
set forth in SEQ ID NO: 4 or having at least 80% identity, more
preferably at least 85% identity, and still more preferably 90%, 92%,
94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4. It is also
provided an isolated nucleic acid sequence from BP 1433 bacteriophage,
or variant thereof. The invention also encompasses isolated polypeptides
encoded by BP 1433 bacteriophage, or variant thereof, or encoded by an
isolated nucleic acid sequence from BP1433 bacteriophage of the
invention. BP1433 bacteriophage of the invention is also characterized
by a PLE below 20, more preferably below 15 and still more preferably of
around 4.
In another particular embodiment, the
invention is related to BP 1450 bacteriophage, or any variant thereof.
BP 1450 bacteriophage, or any variant thereof, can be produced or
expanded in e.g., P. aeruginosa strain PAOl . BP1450, or any variant
thereof, is specific and has lytic activity against LMG24882, LMG24883,
LMG24886, LMG24887, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898,
LMG24903, LMG24904, LMG24905, LMG24909 and/or LMG24913 strains. BP1450
comprises a
genome comprising a sequence as set forth in SEQ ID
NO: 5 or having at least 80% identity, more preferably at least 85%
identity, and still more preferably 90%>, 92%, 94%, 95%, 96%, 97%,
98% or 99% identity to SEQ ID NO: 5. It is also provided an isolated
nucleic acid sequence from BP 1450 bacteriophage, or variant thereof.
The invention also encompasses isolated polypeptides encoded by BP 1450
bacteriophage, or variant thereof, or encoded by an isolated nucleic
acid sequence from BP 1450 bacteriophage of the invention. BP 1450
bacteriophage of the invention is also characterized by a PLE below 20,
more preferably below 15 and still more preferably of around 2.
In
still another aspect, the invention is related to BP 1644
bacteriophage, or any variant thereof. BP 1644 bacteriophage, or any
variant thereof, can be produced or expanded in e.g., P. aeruginosa
strain PAOl . BP1644, or any variant thereof, is specific and has lytic
activity against LMG24882, LMG24883, LMG24886, LMG24891, LMG24892,
LMG24893, LMG24896, LMG24898, LMG24905 and/or LMG24909 strains. BP1644
comprises a genome comprising a sequence as set forth in SEQ ID NO: 6 or
having at least 80%) identity, more preferably at least 85% identity,
and still more preferably 90%>, 92%, 94%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO: 6. It is also provided an isolated nucleic acid
sequence from BP 1644 bacteriophage, or variant thereof. The invention
also encompasses isolated polypeptides encoded by BP 1644 bacteriophage,
or variant thereof, or encoded by an isolated nucleic acid sequence
from BP 1644 bacteriophage of the invention. BP 1644 bacteriophage of
the invention is also characterized by a PLE below 20, more preferably
below 15 and still more preferably of around 1,5.
In
another particular embodiment, the invention is related to BP 1647
bacteriophage, or any variant thereof. BP 1647 bacteriophage, or any
variant thereof, can be produced or expanded in e.g., P. aeruginosa
strain PAOl . BP1647, or any variant thereof, is specific and has lytic
activity against LMG24882, LMG24883, LMG24891, LMG24892, LMG24893,
LMG24896, LMG24898, LMG24903 and/or LMG24916 strains. BP1647 comprises a
genome comprising a sequence as set forth in SEQ ID NO: 7 or having at
least 80%) identity, more preferably at least 85% identity, and still
more preferably 90%>, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to
SEQ ID NO: 7. It is also provided
an isolated nucleic acid
sequence from BP 1647 bacteriophage, or variant thereof. The invention
also encompasses isolated polypeptides encoded by BP 1647 bacteriophage,
or variant thereof, or encoded by an isolated nucleic acid sequence
from BP 1647 bacteriophage of the invention. BP 1647 bacteriophage of
the invention is also characterized by a PLE below 20, more preferably
below 15 and still more preferably of around 0,4.
In
another particular embodiment, the invention is related to BP 1648
bacteriophage, or any variant thereof. BP 1648 bacteriophage, or any
variant thereof, can be produced or expanded in e.g., P. aeruginosa
strain PAOl . BP1648, or any variant thereof, is specific and has lytic
activity against LMG24882, LMG24883, LMG24891, LMG24893, LMG24896,
LMG24898, and/or LMG24909 strains. BP 1648 comprises a genome comprising
a sequence as set forth in SEQ ID NO: 8 or having at least 80%
identity, more preferably at least 85% identity, and still more
preferably 90%>, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ
ID NO: 8. It is also provided an isolated nucleic acid sequence from BP
1648 bacteriophage, or variant thereof. The invention also encompasses
isolated polypeptides encoded by BP 1648 bacteriophage, or variant
thereof, or encoded by an isolated nucleic acid sequence from BP 1648
bacteriophage of the invention. BP 1648 bacteriophage of the invention
is also characterized by a PLE below 20, more preferably below 15 and
still more preferably of around 2.
In another aspect, the
invention is related to BP 1649 bacteriophage, or any variant thereof.
BP 1649 bacteriophage, or any variant thereof, can be produced or
expanded in e.g., P. aeruginosa strain PAOl . BP1649, or any variant
thereof, is specific and has lytic activity against LMG24882, LMG24883,
LMG24886, LMG24887, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898,
LMG24905, LMG24909 and/or LMG24913 strains. BP 1649 comprises a genome
comprising a sequence as set forth in SEQ ID NO: 9 or having at least
80% identity, more preferably at least 85% identity, and still more
preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 9. It is also provided an isolated nucleic acid sequence from BP1649
bacteriophage, or variant thereof. The invention also encompasses
isolated polypeptides encoded by BP 1649 bacteriophage, or variant
thereof, or encoded by an isolated nucleic acid sequence from BP 1649
bacteriophage of the invention. BP 1155 bacteriophage of the invention
is also characterized by a PLE below 20, more preferably below 15 and
still more preferably of around 3,5.
In another
particular embodiment, the invention is related to BP 1650
bacteriophage, or any variant thereof. BP 1650 bacteriophage, or any
variant thereof, can be produced or expanded in e.g., P. aeruginosa
strain PAOl . BP1650, or any variant thereof, is specific and has lytic
activity against LMG24882, LMG24893, LMG24896, LMG24898, LMG24905,
and/or LMG24909 strains. BP1650 comprises a genome comprising a sequence
as set forth in SEQ ID NO: 10 or having at least 80% identity, more
preferably at least 85% identity, and still more preferably 90%, 92%,
94%, 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO: 10. It is also
provided an isolated nucleic acid sequence from BP 1650 bacteriophage,
or variant thereof. The invention also encompasses isolated polypeptides
encoded by BP 1650 bacteriophage, or variant thereof, or encoded by an
isolated nucleic acid sequence from BP 1650 bacteriophage of the
invention. BP 1650 bacteriophage of the invention is also characterized
by a PLE below 20, more preferably below 15 and still more preferably of
around 14.
In still another aspect, the invention is
related to BP 1658 bacteriophage, or any variant thereof. BP 1658
bacteriophage, or any variant thereof, can be produced or expanded in
e.g., P. aeruginosa strain PAOl . BP1658, or any variant thereof, is
specific and has lytic activity against LMG24882, LMG24887, LMG24891,
LMG24893, LMG24896 and/or LMG24898 strains. BP1658 comprises a genome
comprising a sequence as set forth in SEQ ID NO: 11 or having at least
80% identity, more preferably at least 85% identity, and still more
preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID
NO: 11. It is also provided an isolated nucleic acid sequence from
BP1658 bacteriophage, or variant thereof. The invention also encompasses
isolated polypeptides encoded by BP 1658 bacteriophage, or variant
thereof, or encoded by an isolated nucleic acid sequence from BP 1658
bacteriophage of the invention. BP 1658 bacteriophage of the invention
is also characterized by a PLE below 20, more preferably below 15 and
still more preferably of around 3.
In another aspect, the
invention is related to BP 1661 bacteriophage, or any variant thereof.
BP 1661 bacteriophage, or any variant thereof, can be produced or
expanded in e.g., P. aeruginosa strain PAOl . BP1661 or any variant
thereof, is specific and has lytic activity against LMG24882, LMG24883,
LMG24886, LMG24891, LMG24892, LMG24893, LMG24896, LMG24898, and/or
LMG24909 strains. BP1661 comprises a genome comprising a sequence as set
forth in SEQ ID NO: 12 or having at least 80% identity, more preferably
at least 85% identity, and still more preferably 90%>, 92%, 94%,
95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12. It is also provided
an isolated nucleic acid sequence from BP 1661 bacteriophage, or
variant thereof. The invention also encompasses isolated polypeptides
encoded by BP 1661 bacteriophage, or variant thereof, or encoded by an
isolated nucleic acid sequence from BP 1661 bacteriophage of the
invention. BP 1661 bacteriophage of the invention is also characterized
by a PLE below 20, more preferably below 15 and still more preferably of
around 4.
In still another aspect, the invention is
related to BP 1662 bacteriophage, or any variant thereof. BP 1662
bacteriophage, or any variant thereof, can be produced or expanded in
e.g., P. aeruginosa strain PAOl . BP1662 or any variant thereof, is
specific and has lytic activity against LMG24883, LMG24891, LMG24892,
LMG24893, LMG24896, LMG24898, and/or LMG24916strains. BP 1662 comprises a
genome comprising a sequence as set forth in SEQ ID NO: 13 or having at
least 80% identity, more preferably at least 85% identity, and still
more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%) identity to
SEQ ID NO: 13. It is also provided an isolated nucleic acid sequence
from BP 1662 bacteriophage, or variant thereof. The invention also
encompasses isolated polypeptides encoded by BP 1662 bacteriophage, or
variant thereof, or encoded by an isolated nucleic acid sequence from BP
1662 bacteriophage of the invention.
BP 1662 bacteriophage of
the invention is also characterized by a PLE below 20, more preferably
below 15 and still more preferably of around 1.
Nucleic acids and polypeptides
The invention relates to a nucleic acid contained in a bacteriophage of
the invention, or any fragment of such a nucleic acid. The term
fragment designates, more preferably, a fragment containing (or
consisting of) an open reading frame. The nucleic acid may be DNA or
RNA, single- or double-stranded.
The nucleic acid can be
isolated from the deposited bacteriophages, or produced using
recombinant DNA technology (e.g., polymerase chain reaction (PCR)
amplification, cloning), enzymatic or chemical synthesis, or
combinations thereof, according to general techniques known per se in
the art. Also included are homologous sequences and fragments thereof
including, but not limited to, natural allelic variants and modified
nucleic acid sequences in which nucleotides have been inserted, deleted,
substituted, and/or inverted.
In a particular embodiment, the
invention relates to a nucleic acid comprising a sequence selected from
anyone of SEQ ID NOs: 1-13, or a sequence having at least 95%, 96%, 97%,
98%, 99% or more sequence identity to anyone of SEQ ID NOs: 1-13.
In
another particular embodiment, the invention relates to a nucleic acid
comprising the sequence of a fragment of a sequence selected from anyone
of SEQ ID NOs: 1-13, or a fragment of a sequence having at least 95%,
96%, 97%, 98%, 99% or more sequence identity to anyone of SEQ ID NOs:
1-13, said fragment comprising an open reading frame or a regulatory
element such as a promoter.
The nucleic acid of the invention can be in free form, or cloned in a vector.
In
a further aspect, the invention also relates to an isolated polypeptide
encoded by a nucleic acid sequence selected from SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID
NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID
NO: 12 and SEQ ID NO: 13. The polypeptides may be produced by techniques
known per se in the art such as synthesis, recombinant technology, or
combinations thereof. The polypeptides may be isolated or purified, and
used as antibacterial agents or as reagents for in vitro analyses.
Compositions of the invention
One
aspect of the invention relates to compositions comprising at least one
bacteriophage as described above, more preferably at least 2 or more
and, optionally, a pharmaceutically or veterinary acceptable excipient.
As described, the bacteriophages of the invention have very potent
lytic activity against P. aeruginosa strains. Combinations of these
bacteriophages may be produced to expand the host spectrum and produce
highly effective antibacterial compositions.
More
particularly, the invention relates to an antibacterial composition
comprising at least two bacteriophages having lytic activity against a
Pseudomonas aeruginosa (P. aeruginosa) strain, said at least two
bacteriophages being selected from the bacteriophages having a genome
comprising a nucleotide sequence of anyone of SEQ ID NOs: 1 to 13 or a
sequence having at least 90% identity thereto, preferably at least 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
In
a preferred embodiment, the compositions of the invention comprise at
least three, even more preferably at least four distinct bacteriophages
selected from the bacteriophages having a genome comprising a nucleotide
sequence of anyone of SEQ ID NOs: 1 to 13 or a sequence having at least
90% identity thereto, preferably at least 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity thereto. Compositions of the invention may
comprise at least 5, 6, 7, 8, 9, 10, 11, 11, 12 or all of the 13
distinct types of bacteriophages as disclosed above.
One
aspect of the invention is related to a composition at least one
bacteriophage selected from BP1384, BP1429, BP1430, BP1433, BP1450,
BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP1662, or
variants thereof.
The invention also concerns a composition
comprising at least two distinct bacteriophages selected from BP1384,
BP1429, BP1430, BP1433, BP1450, BP1644, BP1647, BP1648, BP1649, BP1650,
BP1658, BP1661 and/or BP1662, or variants thereof.
In a
particular embodiment, a composition of the invention comprises BP 1384
in combination with at least one further bacteriophage selected from
BP1429, BP1430, BP1433, BP1450, or BP1644.
In another particular
embodiment, a composition of the invention comprises BP 1384 in
combination with at least one further bacteriophage selected from BP
1450 and BP 1647.
In another particular embodiment, a
composition of the invention comprises BP1430 in combination with at
least one further bacteriophage selected from BP1450, BP1644, BP1649 and
BP1661.
In another particular embodiment, the
composition comprises BP 1433 in combination with at least one further
bacteriophage selected from BP1450, BP1647, BP1648, BP1650 and BP1658.
In
another preferred embodiment, the composition comprises BP 1384 in
combination with at least one further bacteriophage selected from BP
1429, BP 1647, BP 1649 and BP 1662.
The invention also
relates to a composition comprising a combination of all of the
bacteriophages BP1384, BP1429, BP1430, BP1433, BP1450, BP1644, BP1647,
BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP1662, or variants
thereof.
Specific examples of compositions of the invention comprise:
.
a bacteriophage having a genome comprising a nucleotide sequence of SEQ
ID NO: 1 or a sequence having at least 90% identity thereto, and a
bacteriophage having a genome comprising a nucleotide sequence of SEQ ID
NO: 4 or a sequence having at least 90% identity thereto ;
.
a bacteriophage having a genome comprising a nucleotide sequence of SEQ
ID NO: 1 or a sequence having at least 90% identity thereto, and a
bacteriophage having a genome comprising a nucleotide sequence of SEQ ID
NO: 5 or a sequence having at least 90% identity thereto ;
.
a bacteriophage having a genome comprising a nucleotide sequence of SEQ
ID NO: 3 or a sequence having at least 90% identity thereto, and a
bacteriophage having a
genome comprising a nucleotide sequence of SEQ ID NO: 9 or a sequence having at least 90% identity thereto;
.
a bacteriophage having a genome comprising a nucleotide sequence of SEQ
ID NO: 3 or a sequence having at least 90% identity thereto, and a
bacteriophage having a genome comprising a nucleotide sequence of SEQ ID
NO: 4 or a sequence having at least 90%) identity thereto;
.
a bacteriophage having a genome comprising a nucleotide sequence of SEQ
ID NO: 3 or a sequence having at least 90% identity thereto, and a
bacteriophage having a genome comprising a nucleotide sequence of SEQ ID
NO: 10 or a sequence having at least 90%) identity thereto;
. a
bacteriophage having a genome comprising a nucleotide sequence of SEQ
ID NO: 1 or a sequence having at least 90% identity thereto, and a
bacteriophage having a genome comprising a nucleotide sequence of SEQ ID
NO: 3 or a sequence having at least 90% identity thereto, and a
bacteriophage having a genome comprising a nucleotide sequence of SEQ ID
NO: 4 or a sequence having at least 90%> identity thereto;
.
a bacteriophage having a genome comprising a nucleotide sequence of SEQ
ID NO: 1 or a sequence having at least 90% identity thereto, and a
bacteriophage having a genome comprising a nucleotide sequence of SEQ ID
NO: 3 or a sequence having at least 90% identity thereto, and a
bacteriophage having a genome comprising a nucleotide sequence of SEQ ID
NO: 5 or a sequence having at least 90%> identity thereto; or
.
a bacteriophage having a genome comprising a nucleotide sequence of SEQ
ID NO: 1 or a sequence having at least 90% identity thereto, and a
bacteriophage having a genome comprising a nucleotide sequence of SEQ ID
NO: 3 or a sequence having at least 90% identity thereto, and a
bacteriophage having a genome comprising a nucleotide sequence of SEQ ID
NO: 9 or a sequence having at least 90%> identity thereto.
A specific embodiment of the invention relates to a composition comprising:
. a bacteriophages having a genome comprising a nucleotide sequence of
SEQ ID NO: 1 or a sequence having at least 90% identity thereto;
. a bacteriophages having a genome comprising a nucleotide sequence of
SEQ ID NO: 2 or a sequence having at least 90% identity thereto;
. a bacteriophages having a genome comprising a nucleotide sequence of
SEQ ID NO: 3 or a sequence having at least 90%> identity thereto;
. a bacteriophages having a genome comprising a nucleotide sequence of
SEQ ID NO: 4 or a sequence having at least 90% identity thereto;
. a bacteriophages having a genome comprising a nucleotide sequence of SEQ
ID NO: 5 or a sequence having at least 90%> identity thereto;
. a bacteriophages having a genome comprising a nucleotide sequence of
SEQ ID NO: 6 or a sequence having at least 90% identity thereto;
. a bacteriophages having a genome comprising a nucleotide sequence of
SEQ ID NO: 7 or a sequence having at least 90%> identity thereto;
. a bacteriophages having a genome comprising a nucleotide sequence of
SEQ ID NO: 8 or a sequence having at least 90% identity thereto;
. a bacteriophages having a genome comprising a nucleotide sequence of
SEQ ID NO: 9 or a sequence having at least 90% identity thereto;
. a bacteriophages having a genome comprising a nucleotide sequence of SEQ
ID NO: 10 or a sequence having at least 90% identity thereto;
. a bacteriophages having a genome comprising a nucleotide sequence of
SEQ ID NO: 11 or a sequence having at least 90% identity thereto;
. a bacteriophages having a genome comprising a nucleotide sequence of
SEQ ID NO: 12 or a sequence having at least 90% identity thereto; and
. a bacteriophages having a genome comprising a nucleotide sequence of
SEQ ID NO: 13 or a sequence having at least 90% identity thereto.
The
compositions of the invention may further comprise additional
antibacterial agents, particularly other bacteriophages having distinct
host specificity.
Preferred compositions of the invention are lytic against antibiotic-resistant P. aeruginosa strains.
Further
preferred compositions of the invention are lytic against more that 90%
of all bacterial strains of the LMG collection, obtained from the
well-known BCCM/LMG Bacteria Collection. This collection is accessible
via http://www.cabri.org/CABRI/srs-doc/bccm lmg.info.html web site.
The
antibacterial compositions of the invention may be in various forms,
such as liquid, semi- liquid, solid or lyophilized formulations.
The
compositions of the invention may comprise any effective amount of the
selected bacteriophage(s). Preferably, they comprise between 10e4 and 10e12 PFU of each of said bacteriophages, preferably between 10e5 and 10el°.
PFU. The relative amounts of each type of bacteriophage in a
composition of the invention may be adjusted by a skilled artisan.
Typically, When the antibacterial composition comprises several (n)
distinct bacteriophages as defined above, the total relative amount %A
of each bacteriophage in the composition is more preferably %A=
(100/ni)xV, wherein n; represents the number of distinct types of
bacteriophages and V is a variability factor comprised between 0.2 and
5. Most preferably, V is comprised between 0.3 and 3, even more
preferably between 0.5 and 2, generally between 0.8 and 1.5. In a
preferred typical embodiment, each type of bacteriophage is present in a
composition of the invention in approximately equal relative amounts.
The compositions of the invention preferably comprise a suitable
diluent or carrier, such as a pharmaceutically or veterinary acceptable
excipient or carrier. Compositions according to the present invention
may include any excipient or carrier, such as thickeners, diluents,
buffers, preservatives, surface active agents and the like, in addition
to the bacteriophage(s) of choice. Such includes physiologically
acceptable solutions or vehicles that are harmless or do not cause any
significant specific or nonspecific immune reaction to an organism or do
not abrogate the biological activity of the bacteriophage. For liquid
formulation, saline, sterile water, Ringer's solution, buffered
physiological saline, albumin infusion solution, dextrose solution,
maltodextrin
solution, glycerol, ethanol, and mixtures thereof
may be used as a pharmaceutically or veterinary acceptable excipient or
carrier. If appropriate, other conventional additives such as
thickeners, diluents, buffers, preservatives, surface active agents,
antioxidants and bacteriostatic agents may be added. Further, diluents,
dispersants, surfactants, binders and lubricants may be additionally
added to the composition to prepare injectable formulations such as
aqueous solutions, suspensions, and emulsions, oral formulations such as
pills, capsules, granules, or tablets, or powdered formulations.
Formulations for topical administration may include, band aids,
dressings, patches, films, ointments, lotions, creams, gels, drops,
suppositories, sprays, tampons, sanitary towels, liquids and powders.
Formulations for decontamination or for medical use may also include
aerosols or sprays.
The compositions of the invention may
be used in the medical field, including the human or veterinary medical
areas, for e.g. the treatment of an infection in a mammal or for
improving a subject's condition. The compositions may be used to kill P.
aeruginosa bacteria in an organism, for treating an infection. The
composition may also be used for improving the condition of a mammal by
modifying the microbial flora in said mammal. In particular, the
compositions of the invention can specifically remove P. aeruginosa
strains on the skin or mucous membranes of a mammal, thus modifying its
microbial flora and restoring a proper balance.
In a
particular embodiment, the invention also relates to a method for
treating an infection in a mammal comprising the administration to said
mammal of a composition or bacteriophage or nucleic acid or polypeptide
as defined above. In a particular embodiment the method comprises
administering at least one, preferably at least two, even more
preferably at least three bacteriophages selected from BP 1384, BP 1429,
BP1430, BP1433, BP1450, BP1644, BP1647, BP1648, BP1649, BP1650, BP1658,
BP 1661 and/or BP 1662, or variants thereof.
The invention also
relates to the use of a composition, bacteriophage, nucleic acid or
polypeptide as described for the manufacture of a medicament for
treating an infection in a mammal, or for restoring microbial flora in
said mammal.
The compositions or agents of the invention may be
administered by any convenient route, including intravenous, oral,
transdermal, subcutaneous, mucosal, intramuscular, intrapulmonary,
intranasal, parenteral, rectal, vaginal and topical. In a preferred
embodiment, the bacteriophages or compositions are administered by
topical route, e.g., by application on the skin of a subject. The
compositions may be administered directly or indirectly, e.g., via a
support. In this regard, the compositions may, for example, be applied
or sprayed to the afflicted area. Compositions of the invention can also
be administered by oral or parenteral routes. The dosage suitable for
applying, spraying, or administrating the compositions of the present
invention can be adjusted by the skilled person depending on a variety
of factors including formulation, mode of administration, age, weight,
sex, condition, diet of the mammal being treated at the time of
administration, route of administration, and reaction sensitivity. A
physician having ordinary skills in the art can readily determine and
prescribe the effective amount of the composition required.
The
dosing can also be adjusted by the skilled person so that a lytic
activity against antibiotic-resistant P. aeruginosa strains is obtained.
An efficient dose to obtain a lytic activity in vivo typically includes
a concentration of at least 10e2 PFU/ml, preferably from about 10e2 to 10e12 PFU/ml, depending on the administration route. Administration may be performed only once or, if needed, repeated.
The
compositions of the invention may be administered to treat P.
aeruginosa infections, typically of the respiratory tract, urinary
tract, burns, wounds, ear, skin, or soft tissues, or gastrointestinal or
post-surgical infections.
As shown in the experimental section,
the bacteriophages and compositions of the invention are able to
selectively kill P. aeruginosa bacteria in vitro or in vivo. The
compositions can destroy mixtures of different P aeruginosa bacteria,
even in vivo, even at low dosage. Furthermore, the compositions of the
invention are effective is killing bacteria embedded in biofilms, which
is particularly important for pathogenic bacteria. Also, the
compositions and bacteriophages of the invention are strictly unable to
affect mammalian cells, and are therefore specific and devoid of side
effects in vivo.
The invention also relates to the use of a
composition, bacteriophage, nucleic acid or polypeptide of the invention
for decontaminating a material. Due to their potent antibacterial
effect, and to their ability to even compromise the integrity of a
bacterial biofilm, the compositions of the invention can be used as
decontaminating agent, to eliminate or at least cause a reduction in
bacterial numbers on a material. Such methods may be applied for the
treatment of a variety of biological or non-biological surfaces in both
medical and non-medical contexts, including solid materials or devices
such as, for example, contact lenses, surfaces of devices to be
implanted into the body, pipes, ducts, laboratory vessels, textiles,
etc...
Diagnostic/predictive tests of the invention:
The
invention also concerns a method for predicting or determining the
efficacy of a bacteriophage therapy in a subject, wherein the method
comprises a step of determining a lytic activity of one or more
bacteriophages selected from BP1384, BP1429, BP1430, BP1433, BP1450,
BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP 1662 to
an P. aeruginosa strain from a sample from said subject, such a lytic
activity being indicative of an efficient treatment. In a preferred
aspect, the method further optionally comprises a step of treating said
subject by one or more bacteriophages having a lytic activity to a P.
aeruginosa strain from a sample of said subject.
In
another aspect, the invention provides a method for selecting a subject
or determining whether a subject is susceptible to benefit from a
bacteriophage therapy, wherein the method comprises the step of
determining a lytic activity of one or more bacteriophages selected from
BP1384, BP1429, BP1430, BP1433, BP1450, BP1644, BP1647, BP1648, BP1649,
BP1650, BP1658, BP1661 and/or BP1662 to an P. aeruginosa strain from a
sample of said subject, a lytic activity of one or more bacteriophages
of the invention to at least one P. aeruginosa strain indicating a
responder subject.
Another object of the invention
relates to a method for predicting the response of a subject to a
bacteriophage therapy, wherein the method comprises the step of
determining a lytic activity of one or more bacteriophage selected from
BP1384,
BP1429, BP1430, BP1433, BP1450, BP1644, BP1647, BP1648,
BP1649, BP1650, BP1658, BP1661 and/or BP1662 to a P. aeruginosa strain
from a sample of said subject, a lytic activity of one or more
bacteriophage of the invention to at least one P. aeruginosa strain
being indicative of a good response to said therapy.
Further
aspects and advantages of the invention will be disclosed in the
following experimental section, which is illustrative only.
EXAMPLES
MATERIALS AND METHODS
Phage isolation and preparation
MDR P. aeruginosa bacteria were used for isolating and enriching each
virulent bacteriophage from environmental water. Environmental samples
and overnight culture of bacteria in Luria Bertani (LB) were mixed and
incubated at 37°C for 24h with shaking to enrich specific
bacteriophages. At the end of incubation, drops of chloroform were added
to the culture. The culture was spun down at 11,000 g for 5 minutes to
remove bacterial cells and debris. The supernatant was subjected to 0.2
μιη filter to remove the residual bacterial cells. The enriched phage
solution was plated on LB agar medium with P. aeruginosa embedded.
Plaques formed on the plates after 24h incubation at 37°C. Single plaque
was picked out for subsequent phage purification and amplification. The
phage was then stored at 4°C in a suspension in LB broth or
physiological saline.
The titer of phage in a suspension was
estimated by plaque counting (Postic, 1961). 10-fold dilutions of a
suspension were delivered on a dried lawn of the propagating strain. The
plates were read after overnight incubation. The plaque-counting method
also permitted visualization of plaque morphology.
Host range determination.
The host ranges of bacteriophages were determined among a collection of 20 P. aeruginosa from the LMG collection. 109 bacterial cells were mixed with melted agar and this mixture was poured on solid agar to make double layer agar plates. After
solidification,
isolated bacteriophage stock solutions were spotted on each plate with
different bacterium strain. After allowing 20 min for the spots to be
absorbed, the plates were inverted and incubated for 24h at 37°C before
the degree of lysis was recorded (Postic, 1961; Yang, 2010).
Electron microscopy.
Electron micrographs of each phage were taken with a transmission electron microscope.
Sequencing, analysis and annotation of phage genomes.
To isolate phage DNA, phages were propagated as described above. Phage
DNA was isolated by extraction with phenol:chloroform:isoamyl alcohol
(25:24: 1, V/V), ethanol precipitation and resolution in water. Whole
genome sequencing was done and the BLAST algorithm was used to determine
the similarity to described genes in the National Center for
Biotechnology Information [NCBI] database. The genomes were scanned for
potential open reading frames (ORFs).
EXAMPLE 1 : Bacteriophage-host characteristics and kinetics
One-step
growth experiments were carried out according to the previous
descriptions to determine first the productive lytic time, adsorption
rate, and then the phage burst size. To determine the adsorption rate
samples were taken at different time intervals to analyze the free phage
particles in the solutions. For productive time and phage burst size
determination, P. aeruginosa bacteria were mixed with phages solutions
and phages were allowed to adsorb for 15 min. The mixture was subjected
to centrifugation immediately at 5000 rpm for 10 min to remove free
phage particles. The pellet was resuspended in 5 fresh LB medium and the
culture was continuously incubated at 37°C. Samples were taken at 3 min
intervals and phage titre was determined. These results permitted to
calculate the number of phages produced per bacteria (burst size), the
productive time and the productive lytic effect (PLE), as shown in table
5 below.
Table 4
These
results show that all phages have potent viral production capacity and
absorption rates. Most phages have a PLE below 7, which demonstrates a
remarkable profile. Phages 1429 and 1647 are particularly effective in
this regard. In addition, the different PLE and adsorption times permit
to create cocktails with selected variability.
EXAMPLE 2: Preparation of cocktail compositions
The following cocktail compositions are constituted, each comprising between 10-9 and 10-11 pfu of each bacteriophage:
Table 5
The
following additional two cocktail compositions comprising all of the
various phages are constituted, covering the most important diversity of
P. aeruginosa species:
Cocktail composition A :
Phage 1384 1429 1430 1433 1450 1644 1647 1648
titer 4,00E+10 l,23E+09 5,45E+08 8,33E+10 8,91E+10 9,09E+08 2,00E+09 3,09E+09
Phage 1649 1650 1658 1661 1662
Titre 9,00E+09 9,45E+08 l,91E+09 l,14E+09 3,55E+08
Cocktail composition B
phage 1649 1650 1658 1661 1662
Titre 1,00E+11 2,20E+11 8,00E+10 1,00E+11 6,00E+07
EXAMPLE 3: Sensitivity of bacteria to bacteriophage cocktails of the invention
Various strains of bacteria were tested with the bacteriophage cocktails of the invention at 2.109 bacteriophages/ml. Different bacterial concentrations were plated on the bacteriophage cocktail at 2.109 bacteriophages/ml and incubated 24h at 37°C.
Cocktails
are tested on the 22 distinct P. aeruginosa bacteria listed in tables 2
and 3. The % of bacteria species sensitive to the cocktails are listed
in table 6 below:
Table6
Bacteria
were enumerated and used to the calculation of resistance rate (number
of bacteria after incubation/number of bacteria plated). Resistance
rates with a cocktail comprising the 13 different types of
bacteriophages are shown in the following table 7:
Table 7
All tested bacteria are sensitive to compositions of the invention.
EXAMPLE 4: Cocktail specificity
The
cocktail specificity was confirmed by testing on ten bacteria species,
including Escherichia coli, Acinebacter baumanii, Enterobacter aerogenes
C, Enterobacter asburiae, Enterobacter cloacae, Klebsiella pneumoniae,
Proteus mirabilis, Staphylococus aureus, Stenotrophomonas maltophila,
Serratia marcescens.
Table 8 summarizes lytic activity
observed for each bacteriophage used independently or in combination as a
cocktail of 13 bacteriophages.
The above table clearly show that no lytic activity on bacteria other
than P. aeruginosa strain occurred. The bacteriophages and cocktail of
the invention are therefore highly specific for P. aeruginosa strains.
EXAMPLE 5: Efficiency of bacteriophages on P. aeruginosa strain in vitro
Several
strains of the LMG collection were chosen to represent the genetic
diversity of P. aeruginosa and various forms of antibiotic resistance.
Strains were either sensitive or resistant to one or several
antibiotics, as described in Table 9. They were grown individually or in
combination with 2 to 8 strains. The bacteriophage cocktail was added
at a MOI of 1 to 10e"6 , i.e. at a dilution ratio (bacteria/phage) of 1 to 1 million.
Table 9: information about the bacterial strains
The results are presented in Fig 1 and in the following Table 10.
Table 10: Efficiency of bacteriophage cocktail obtained in vitro on P. aeruginosa mixture: at 2.10e7 cfu/ml and at various dilutions:
The
compositions of the invention are able to kill a mixture of 8 distinct
strains of P. aeruginosa bacteria together. The cocktail remains
efficient against 8 strains at a dilution of 1/1000.
EXAMPLE 6: Efficiency of bacteriophages on P. aeruginosa strain in vivo
An isolated Is580 strain, collected on a burned patient on 1997, was used for the following experiments.
Is580 strain is resistant to ampicillin, AMC, PIP, CEF, CXM, axetil CXM, FOX, CPD, CTX, CAZ, GEN, TOB, OFX, NIT, SXT.
SKH1 mouse (or hairless mouse) was used as mouse model of P. aeruginosa infection.
Modus operandi : (see table 1 1 below)
Mice were immunodepressed by 3 IP injections of 1.5 mg of cyclophosphamide
(Cy), every 2 days from the Day -3 before infection.
Mice were burned on skin by 2μ1 of liquid yperite at 30 mg/kg.
Infection two days after the burn by subcutaneous injection of a bacterium suspension in burned site.
Table 1 1 :
Day -3 -2 -1 0 1
l,5mg l,5mg Cy Burn l,5mg Cy Infection
Cy
Injection
IP Yperite IP SC 107cfU IP route
SC injection of cocktail
PHAGE (ΙΟΟμΙ, i.e.108 PFU)
6h post- infection
Cocktail compositions were prepared according to example 1 and compresses soaked of bacteriophages cocktail at 10e7 phages/ml were applied at Day 0.
Various concentrations of P. aeruginosa strains were tested with ΙΟΟμΙ
of bacteriophage cocktail. As shown on Figure 2, all P. aeruginosa
strains were killed 6h post-treatment.
Upon
administration of Is580 P. aeruginosa strain by sub-cutaneous injection
to SKH1 mice, only 35% of mice survived in the absence of further
treatment. In the mice treated by injection of a bacteriophage cocktail
as presented in table 10 above, a remarkable survival rate was observed
(see Fig 3): 100% survival for SKH1 mice treated subcutaneously, 16 days
after infection. By comparison, a 50%> survival was observed for
SKH1 mice treated by antibiotic 2 days after infection.
Accordingly, the compositions of the invention can treat an infection in
vivo and can induce a 100% survival rate in infected mice.
REFERENCES
Clark WA, 1962, Appl Microbiol. Comparison of several methods for preserving bacteriophages.1962 Sep; 10:466-71.
Drulis-Kawa
Z, Majkowska-Skrobek G, Maciejewska B, Delattre AS, Lavigne R, 2012,
Learning from bacteriophages - advantages and limitations of phage and
phage-encoded protein applications. ;13(8):699-722.
Fordos J.
1859. Receuil des travaux de la Societe d'Emulation pour les Sciences
Pharmaceutiques, vol 3 Societe d'Emulation pour les Sciences
Pharmaceutiques, Paris, France
Freeman L. 1916. Chronic general infection with the Bacillus pyocyaneus. Ann. Surg. 64: 195-202.
Gang RK, Bang RL, Sanyal SC, Mokaddas E, Lari AR. 1999. Pseudomonas aeruginosa septicaemia in burns. Burns 25:611-616.
Jones AM, et al. 2010. Clinical outcome for cystic fibrosis patients
infected with transmissible Pseudomonas aeruginosa: an 8-year
prospective study. Chest 137: 1405-1409.
Kang CI, et al.
2005. Bloodstream infections caused by antibiotic-resistant
gram-negative bacilli: risk factors for mortality and impact of
inappropriate initial antimicrobial therapy on outcome. Antimicrob.
Agents Chemother. 49:760-766.
Micek ST, et al. 2005.
Pseudomonas aeruginosa bloodstream infection: importance of appropriate
initial antimicrobial treatment. Antimicrob. Agents Chemother. 49:
1306-1311.
Strateva T. and Yordanov D. 2009. Pseudomonas
aeruginosa - a phenomenon of bacterial resistance. Journal of Medical
Microbiology 58, 1133-1148.
Weinbauer MG. Ecology of prokaryotic viruses. FEMS Microbiol Rev 2004; 28: 127-81.
Williams
EP, Cameron K. 1894. Infection by the Bacillus pyocyaneus a cause of
infantile mortality. Public Health Pap. Rep. 20:355-360.
|
