U.S. patent application number 15/614420 was filed with the patent office on 2018-03-01 for bacteriophage-containing therapeutic agents.
The applicant listed for this patent is BIOCONTROL LIMITED. Invention is credited to David HARPER, Catherine HAWKINS, James SOOTHILL.
Application Number | 20180055895 15/614420 |
Document ID | / |
Family ID | 34105984 |
Filed Date | 2018-03-01 |
United States Patent
Application |
20180055895 |
Kind Code |
A1 |
SOOTHILL; James ; et
al. |
March 1, 2018 |
BACTERIOPHAGE-CONTAINING THERAPEUTIC AGENTS
Abstract
The present invention relates in its broadest aspect to combined
phage/antibiotic therapy. More particularly, it relates to use of
(i) one or more bacteriophages and (ii) one or more antibiotics in
the manufacture of a combined product for simultaneous, separate or
sequential administration of (i) and (ii) to treat a bacterial
infection characterized by biofilm formation, for example an
infection comprising or consisting of P. aeruginosa. Treatment in
this context may be either therapeutic or prophylactic treatment.
Also provided are deposited bacteriophages each exhibiting
different strain specificity against P. aeruginosa and combinations
of such bacteriophages, e.g. a panel of six deposited
bacteriophages which was found to be effective against a high
percentage of clinical isolates of P. aeruginosa from canine ear
infections.
Inventors: |
SOOTHILL; James; (London,
GB) ; HAWKINS; Catherine; (London, GB) ;
HARPER; David; (Southampton, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOCONTROL LIMITED |
Southampton |
|
GB |
|
|
Family ID: |
34105984 |
Appl. No.: |
15/614420 |
Filed: |
June 5, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13757655 |
Feb 1, 2013 |
9687514 |
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15614420 |
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13333684 |
Dec 21, 2011 |
8388946 |
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13757655 |
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12897741 |
Oct 4, 2010 |
8105579 |
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13333684 |
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10565347 |
Jul 12, 2006 |
7807149 |
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PCT/GB04/03237 |
Jul 23, 2004 |
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12897741 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/1214 20130101;
A61P 43/00 20180101; C12N 2795/00032 20130101; A61K 45/06 20130101;
A61P 27/16 20180101; A61P 31/00 20180101; A61K 31/424 20130101;
A61P 27/02 20180101; C12N 2795/00031 20130101; A61P 31/04 20180101;
C07K 14/21 20130101; A61P 11/00 20180101; A61P 17/02 20180101; A61P
27/04 20180101; A61K 38/51 20130101; A61K 35/76 20130101; A61K
31/43 20130101; A61K 38/00 20130101; A61K 35/76 20130101; A61K
2300/00 20130101 |
International
Class: |
A61K 35/76 20060101
A61K035/76; A61K 45/06 20060101 A61K045/06; A61K 31/43 20060101
A61K031/43; A61K 38/51 20060101 A61K038/51; A61K 31/424 20060101
A61K031/424 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 23, 2003 |
GB |
0317240.0 |
May 14, 2004 |
GB |
0410855.1 |
Claims
1-25. (canceled)
26. A method of therapeutic or prophylactic treatment of a
bacterial infection characterised by biofilm formation which
comprises administering to a human or non-human animal in need
thereof one or more bacteriophages capable of targeting bacteria of
said infection and simultaneously, separately or sequentially
thereto one or more antibiotics.
27. The method of claim 26 wherein the one or more bacteriophages
are selected from NCIMB 41174, NCIMB 41175, NCIMB 41176, NCIMB
41177, NCIMB 41178, NCIMB 41179, NCIMB 41180 and NCIMB 41181
(deposited at the National Collection of Industrial and Marine
Bacteria), and mutants thereof which retain the ability to target
P. aeruginosa.
28. canceled
29. A non-therapeutic method of removing, reducing or preventing
bacterial contamination characterised by biofilm formation, said
method comprising applying to the site or prospective site of said
contamination one or more bacteriophages capable of targeting
bacteria of said contamination and simultaneously, separately or
sequentially thereto one or more antibiotics or antiseptics.
30. The method of claim 29 wherein one or more bacteriophages are
selected from NCIMB 41174, NCIMB 41175, NCIMB 41176, NCIMB 41177,
NCIMB 41178, NCIMB 41179, NCIMB 41180 and NCIMB 41181 (deposited at
the National Collection of Industrial and Marine Bacteria), and
mutants thereof which retain the ability to target P.
aeruginosa.
31. A non-therapeutic method of removing, reducing or preventing
bacterial contamination comprising or consisting of P. aeruginosa,
said method comprising applying to the site or prospective site of
said contamination one or more bacteriophages selected from NCIMB
41174, NCIMB 41175, NCIMB 41176, NCIMB 41177, NCIMB 41178, NCIMB
41179, NCIMB 41180 and NCIMB 41181 (deposited at the National
Collection of Industrial and Marine Bacteria), and mutants thereof
which retain the ability to target P. aeruginosa.
32. A method of detecting the presence of P. aeruginosa in an in
vitro sample, which comprises contacting said sample with one or
more bacteriophages selected from NCIMB 41174, NCIMB 41175, NCIMB
41176, NCIMB 41177, NCIMB 41178, NCIMB 41179, NCIMB 41180 and NCIMB
41181 (deposited at the National Collection of Industrial and
Marine Bacteria), and mutants thereof which retain the ability to
target P. aeruginosa and determining whether said bacteriophage(s)
are capable of killing bacteria in said sample.
33. A method of identifying a bacterial strain selective for one of
the bacteriophages NCIMB 41174, NCIMB 41175, NCIMB 41 176, NCIMB 41
177, NCIMB 41178, NCIMB 41179, NCIMB 41 180 and NCIMB 41181, the
method comprising the steps of measuring plaque formation by said
bacteriophage in a number of bacterial strains and selecting a
strain which allows at least 1000 times more plaque formation by
said bacteriophage than by any of said other bacteriophages.
34. A bacterial strain identified by the method of claim 33.
35. Use of one or more bacterial strains according to claim 34 to
identify and/or quantify bacteriophages present in preparations
intended for therapeutic use and/or to identify strains present in
tissue samples obtained during such therapeutic use or following
such use.
36. The method of claim 26 comprising administering a plurality of
bacteriophages capable of infecting the same bacterial species,
each member of said plurality of bacteriophages having a different
strain specificity.
37. The method of claim 26, wherein the one or more antibiotic
is/are administered after said one or more bacteriophages.
38. The method of claim 26, wherein said bacterial infection
comprises or consists of Pseudomonas aeruginosa.
39. The method of claim 26, wherein the method is a method of
prophylactic treatment.
40. The method of claim 26 further comprising administering an
alginase.
41. The method of claim 26, wherein said one or more bacteriophages
are comprised in a contact lens solution or additive.
42. The method of claim 41, wherein the contact lens solution or
additive further comprises one or more antibiotics.
43. The method of claim 26, wherein said infection is an infection
selected from the group consisting of infection of a skin burn or
other skin wound, a lung infection, an ocular infection, and an ear
infection.
44. The method of claim 43, wherein said infection is a canine ear
infection.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to therapeutic and diagnostic
preparations comprising viruses that kill bacteria
(bacteriophages). In particular, the invention provides in a
preferred aspect therapeutic compositions comprising combinations
of bacteriophages as control agents for infections of animals and
humans caused by pathogenic bacteria of the species Pseudomonas
aeruginosa. The invention also relates to use of bacteriophages in
combination with antibiotics to treat bacterial infections
characterised by biofilm formation, especially for example such
infections comprising infection with Pseudomonas aeruginosa such as
canine ear infections
BACKGROUND TO THE INVENTION
[0002] Antibiotics have been seen for many years as "the answer" to
the problem of bacterial infections. This attitude persisted until
the development of the wide-ranging (and in some cases total)
resistance to antibiotics seen within the last ten years. In many
cases it is necessary to use expensive "drugs of last resort" (such
as vancomycin for Staphylococcus aureus), which often require
complex routes of administration and show toxic side effects,
necessitating prolonged hospital treatment.
[0003] Even to these drugs, resistance is reaching worrying levels.
It is now clear that bacteria can adapt to resist any antibiotic.
Even the new generation drugs such as linezolid are already
generating resistance [Mutnick et al (2003) An. Pharmacother.
37:769-774; Rahini et al (2003) Clin Infect Dis 36: E146-148], and
it is clear from recent developments that resistance develops
faster than new antibiotics can be produced, evaluated and
processed through regulatory approvals.
[0004] A further disadvantage of antibiotic treatment is its lack
of specificity. Antibiotics can kill a wide range of bacteria and
this can lead to recolonisation of the body by inappropriate and
often harmful bacteria. There is therefore need for antibacterial
treatments that show specificity against particular bacterial
species so that little resistance is induced in the normal
flora.
[0005] The need for new forms of antibacterial therapy is well
illustrated by the case of infection with the gram-negative aerobic
bacterium Pseudomonas aeruginosa.
[0006] Pseudomonas aeruginosa is a serious opportunistic bacterial
pathogen. Infections caused by Pseudomonas aeruginosa include:
[0007] Otitis externa and otitis media in dogs, ear infections
which exemplify biofilm-based colonization of a body surface and
which are common in inbred (pedigree) dogs; [0008] Otitis externa
of humans ("swimmers ear") along with other ear infections and
other topical infections of humans including Pseudomonas keratitis
and Pseudomonas folliculitis; [0009] Infection of burns and skin
grafts in humans; [0010] Hospital-acquired infections; [0011] Lung
infection in cystic fibrosis (CF) patients.
[0012] 10-15% of nosocomial (hospital acquired) infections are due
to Pseudomonas aeruginosa, with 2 million cases annually in the
U.S. alone. In some situations, the frequency is even higher. Of
around 150,000 burn patients treated in U.S. hospitals and bum
centres per year, 26% have Pseudomonas aeruginosa infections.
Pseudomonas aeruginosa is notorious for its resistance to
antibiotics so infections caused by it can be difficult to treat.
One of its natural habitats is soil, where it is exposed to
organisms that produce antibiotics. This may well have led to the
development of resistance mechanisms coded for both by genes on the
chromosome and by transferable genetic elements known as plasmids.
The properties of the P. aeruginosa outer membrane are important in
conferring resistance. An additional resistance mechanism is its
tendency to grow on available surfaces as complex layers known as
biofilms [Donlan (2002) Emerging Infectious Diseases 8: 881-890,
http://www.cdc.gov/ncidod/EID/vol8no9/02-0063.htm; Fletcher &
Decho (2001) Biofilms in Encyclopaedia of Life Sciences, Nature
Publishing, London; http://www.els.net] that are resistant to far
higher concentrations of antibiotics than are required to kill
individual cells [Chen et al (2002) Pseudomonas infection;
http://www.emedicine.com/PED/topic2701.htm; Qarah et al (2001)
Pseudomonas aeruginosa infections;
http://www.emedicine.com/MED/topic1943.htm; Todar K. (2002) Todar's
Online Textbook of Bacteriology: Pseudomonas aeruginosa;
http://textbookofbacteriology.net/pseudomonas.html; Iglewski B. H
(1996) Pseudomonas. Medical Microbiology 4th edition, S. Baron
(ed.). University of Texas;
http://gsbs.utmb.edu/microbook/ch027.htm]. The practical effect of
this is demonstrated by infections in cystic fibrosis patients,
virtually all of whom eventually become infected with a bacterial
strain that cannot be eradicated by the use of antibiotics, even
when the isolated strain may appear to be sensitive in the
laboratory [Hoiby N (1998) Pseudomonas in cystic fibrosis: past,
present, future. European Cystic Fibrosis Society Joseph Levy
Memorial Lecture;
http://www.ecfsoc.org/pa_review/nh_lect.html].
[0013] Pseudornonas aeruginosa expresses a range of genes (most
notably the algC gene) which produce the extracellular components
responsible for biofilm formation, which are often polysaccharide
in nature (Friedman and Kolter, Mol. Microbiol. (2004) 3, 675-690).
Such biofilm formation is now known to be a characteristic of many
important pathogenic bacteria contributing to increased resistance
to antibiotics. Such biofilms may comprise more than one type of
bacterium supported and surrounded by an excreted extracellular
matrix and assist bacteria to colonise surfaces from marine reefs
to teeth enamel. Biofilms allow bacteria to attach to surfaces and
to attain population densities which would otherwise be
unsupportable. They impart increased resistance to not only
antibiotics but many environmental stresses including toxins such
as heavy metals, bleaches and other cleaning agents. It was
previously thought that contribution of biofilm formation to
antibiotic resistance was primarily a physical process arising from
limitation of diffusion, but more recent evidence has shown that
some biofilms appear to have specific abilities to trap antibiotics
(Mah et al., Nature (2003) 426, 306-310). 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
single-celled ("planktonic") forms. This 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 colonisation once antibiotics are no longer
present. It is clear therefore that biofilms are major factors in
many human diseases.
[0014] Chemical treatments are unsuited to use against biofilms
since this is precisely what they have evolved to counter and many
surfaces where biofilms aid bacterial pathogenesis are poorly
suited to rigorous abrasion. Physical abrasion does provide a means
to disrupt biofilms. However, many surfaces where biofilms aid
bacterial pathogenesis are poorly suited to rigorous abrasion. For
example, the surfaces of wounds or bums 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 biofilin and is partially cleared by regular brushing.
However, bacteria are maintained on unbrushed surfaces (for example
in the gaps between teeth) and can recolonise cleared surfaces both
rapidly and effectively. From this, it is clear that existing
approaches to clearing biofilms are of limited efficacy.
[0015] In addition to the biofilm problem, only a few antibiotics
in any case are capable of effective action against Pseudomonas
aeruginosa, including fluoroquinolones, gentamicin and imipenem,
and even these antibiotics are not effective against all strains.
Multidrug resistance is common and increasing [Friedland I et al
(2003). Diagnostic Microbiology and Infectious Disease 45:245-50;
Henwood et al (2001). Journal of Antimicrobial Chemotherapy 47:
789-799]. The U.S. National Nosocomial Infections Surveillance
System Report of June 1999 [Gerberding J et al (2001). National
Nosocomial Infections Surveliance (NNIS) System Report, data
summary from January 1992-June 2001, issued August 2001. U.S.
Department of Health and Human Services, Atlanta,
http://www.cdc.gov/ncidod/hip/NNIS/2001nnis_report.PDF] states that
antibiotic resistance of Pseudomonas aeruginosa isolated from
nosocomial infections in ICU patients in 1999 had increased over
the 1994-98 period for all classes of antibiotics studied. There is
therefore a demonstrated need for new approaches to the control of
Pseudomonas aeruginosa infection. The inventors in this instance
have addressed this problem through use of new bacteriophage-based
therapies.
[0016] Bacteriophages (often known simply as "phages") are viruses
that grow within bacteria. The name translates as "eaters of
bacteria" and reflects the fact that as they grow most
bacteriophages kill the bacterial host as the next generation of
bacteriophages is released. Early work with bacteriophages was
hindered by many factors, one of which was the widespread belief
that there was only one type of bacteriophage, a nonspecific virus
that killed all bacteria. In fact, the host range of bacteriophages
(the spectrum of bacteria they are capable of infecting) is often
very specific. This specificity may be considered a therapeutic
strength as populations of bacteriophages can be selected to
specifically eliminate only the target bacteria. Antibiotics, on
the other hand, kill a wide range of bacteria and their use can
consequently lead to disruption of the normal flora, leading to
recolonisation of the body by inappropriate and often harmful
bacteria.
[0017] Despite the therapeutic advantages afforded by the host
specificity of bacteriophages, this characteristic has the
disadvantage that it can be difficult to achieve breadth of
coverage of target strains. For this reason, there has been
interest in finding combinations of bacteriophages having broad
targeting capability in relation to particular types of bacterial
infection (see for example Pirsi, The Lancet (2000) 355,1418)
[0018] The inventors in this instance have established a
combination of bacteriophages consisting of six bacteriophages each
with a different strain specificity against Pseudomonas aerguinosa
and which is particularly suitable for broad targeting of P.
aerguinosa infections, especially, for example, canine ear
infections. The combination was found to be capable of destroying
90% of P. aeruginosa strains sampled from canine otitis externa and
other canine ear infections. Furthermore, they have established
that such a phage combination may be employed synergistically with
antibiotic treatment to gain improved efficacy. As a consequence,
it is now extrapolated that combined phage/antibiotic therapy
represents a new general advantageous approach for tackling
bacterial infections characterised by biofilm formation.
[0019] Phage and antibiotic therapy have previously been used
together in Eastern Europe (see for example Bradbury, The Lancet
(February 2004) 363, 624-625), but there was no specific relation
to biofilm formation. Additionally, there have been suggestions
that antibiotics can have adverse effect on use of bacteriophage
therapy since bacteriophages use bacterial metabolism to replicate
and thisis inhibited by antibiotics (Payne and Janssen, Clinical
Pharmacokinetics (2002) 42, 315-325).
SUMMARY OF THE INVENTION
[0020] In one aspect, the present invention thus provides the use
of (i) one or more bacteriophages and (ii) one or more antibiotics
in the manufacture of a combined product for simultaneous, separate
or sequential administration of (i) and (ii) to treat a bacterial
infection characterized by biofilm formation, e.g. a bacterial
infection comprising or consisting of Pseudomonas aeruginosa.
[0021] Treatment of such a bacterial infection in this context will
be understood to mean either therapeutic treatment or prophylactic
treatment. Bacteriophages are uniquely suited to prophylactic use
because: [0022] Chemical agents must be used above specific minimum
levels if they are to be effective. Lower levels are at best
ineffective. At worst, they can encourage the development of
resistance. [0023] Replicating biological agents in contrast have
the innate ability to generate therapeutic dose as and when needed,
even from a very low input dose
[0024] The present invention also provides a panel of
bacteriophages active against Pseudomonas aeruginosa each
exhibiting a different strain specificity. More particularly, the
invention provides eight bacteriophages deposited at the National
Collection of Industrial and Marine Bacteria, Aberdeen, U.K. on
24th Jun. 2003 as NCIMB 41174, NCIMB 41175, NCIMB 41176, NCIMB
41177, NCIMB 41178, NCIMB 41179, NCIMB 41180 and NCIMB 41181 and
mutants thereof which retain the ability to target P. aeruginosa.
While members of the panel might be used individually, use of
combinations of such phages is preferred so as to broaden target
strain efficacy. As indicated above, a combination of six of these
phages, more particularly N41174 to N41179, has been found to be
particularly advantageous in treating canine ear infections
comprising P. aeruginosa and might also he advantageously employed
in treating other P. aeruginosa infections, especially in
combination with antibiotic treatment. Such phage treatment or
combined phage and antibiotic treatment may also be combined with
use of alginase. Again, such treatment will be understood to
encompass prophylactic treatment.
[0025] The invention also extends to non-therapeutic methods of
removing, reducing or preventing bacterial contamination
characterised by biofilm formation. In one embodiment, such a
method comprises applying to the site or prospective site of
contamination one or more bacteriophages capable of targeting
bacteria of the contamination and simultaneously, separately or
sequentially thereto one or more antibiotics or antiseptics. in
another embodiment, there is provided a method of removing,
reducing or preventing bacterial contamination comprising or
consisting of P. aeruginosa which comprises applying to the site or
prospective site of contamination one or more of the deposited
bacteriophages noted above.
[0026] The phages of the invention may also be used in methods for
detecting the presence of target P. aerguinosa strains.
Accordingly, the invention provides a method of detecting P.
aeruginosa in an in vitro sample, e.g. a biological sample from a
human or animal for diagnostic purpose, comprising contacting said
sample with one or more bacteriophages of the invention, and
determining whether said bacteriophage(s) are capable of killing
bacteria in said sample.
[0027] The invention also provides a method of identifying a
bacterial strain indicative for a bacteriophage selected from the
eight deposited bacteriophages listed above, the method comprising
the steps of measuring plaque formation by the bacteriophage in a
number of bacterial strains and selecting a strain which allows at
least 1000 times more plaque formation by said bacteriophage than
by any other of said deposited bacteriophages.
[0028] Also provided are bacterial strains identified by such a
method that can be used to identify bacteriophages present in
preparations intended for therapeutic use and/or to identify
strains present in tissue samples obtained during such therapeutic
use or following such use. Such bacterial strains may also be used
as count strains to determine the amount of a particular
bacteriophage capable of infecting the strain in a bacteriophage
preparation.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIGS. 1A-1D:Efficacy of bacteriophages against different
strains of Pseudomonas aeruginosa. Strains named in bold were
resistant. [0030] .quadrature. Plaques observed [0031] No plaques
[0032] Either (1) Some dilutable inhibition observed but no obvious
plaques, or (2) by visual assessment P.aeruginosa isolate deemed
poorly susceptible [0033] Not done
[0034] The six bacteriophages BC-BP-01, BC-BP-02, BC-BP-03,
BC-BP-04, BC-BP-05, and BC-BP-06 (corresponding to deposits NCIMB
41174, NCIMB 41175, NCIMB 41176, NCIMB 41177, NCIMB 41178 and NCIMB
41179 respectively) together resulted in 90% coverage of all
screened P. aeruginosa strains.
Examples of bacterial isolates used:
TABLE-US-00001 Species Date Number of Bacteria Strain origin
isolated Location passages 7 Pseudomonas Human 1960's US army
surgical 10-100 Used for aeruginosa research unit Ft Sam BC-BP-04
Houston, Texas, USA 3708 Pseudomonas Human 1970's Public Health
10-100 Used for aeruginosa Laboratory, Cambridge, BC-BP-01 UK G184
Pseudomonas Human 1980's Edinburgh, UK 10-100 Used for aeruginosa
BC-BP-02 919686 Pseudomonas Dog 1980's Idexx 2-3 Used for
aeruginosa Laboratories, BC-BP-05 Wetherby, UK 27225 Pseudomonas
Dog 2003 Royal Veterinary 2-3 Used for aeruginosa College, London,
UK BC-BP-06 C33138 Pseudomonas Dog 2003 Axiom laboratories, 2-3
Used for aeruginosa Devon, UK BC-BP-03
[0035] FIG. 2: identification of a BC-BP-03 count strain. Plates of
count strain infected as follows: [0036] A: Uninfected. [0037] B:
Infected with BC-BP-03 (1,000,000-fold dilution). [0038] C:
Infected with BC-BP-01 (10-fold dilution). [0039] D: Infected with
BC-BP-04 (10-fold dilution). [0040] E: Infected with BC-BP-02 (10
fold dilution).
[0041] FIG. 3: Resolution of infection in a dog ear treated with
bacteriophage BC-BP-04: [0042] A: Appearance of right ear 24 hours
after treatment with 400 infectious units of BC-BP-04. [0043] B:
Appearance of left ear which did not receive bacteriophage
treatment.
[0044] FIG. 4: Improvement in total clinical score as % of initial
level (occlusion, erythema, ulceration, discharge type, discharge
volume, odour) of six dogs with antibiotic resistant otitis after 2
days treatment with a combined bacteriophage preparation containing
the six bacteriophages NCIMB 41174 to NCIMB 41179 (BioVet-PA)
(thicker continuous line is average)
[0045] FIG. 5: Pseudomonas bacteria count per gramme of detritus as
% of initial level in the same dog treatment group after 2 days
treatment (thicker continuous line is average)
[0046] FIG. 6: Number of bacteriophages per gramme of detritus as %
of original level (log scale) in the same dog treatment group after
2 days of treatment (thicker continuous line is average)
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present invention utilises panels of naturally-occurring
viruses that infect pathogenic bacteria. Such panels can be
formulated into therapeutic medicaments suitable for evaluation
through the clinical trials process. As indicated above, in one
aspect, the invention provides use of (i) one or more
bacteriophages and (ii) one or more antibiotics in the manufacture
of a combined product for simultaneous, separate or sequential
administration of (i) and (ii) to treat a bacterial infection
characterised by biofilm formation. For this purpose, a panel of
two or more bacteriophages may be employed in the manufacture of a
single combined bacteriophage preparation. The bacteriophages of
the chosen panel will preferably be capable of infecting the same
bacterial species and each exhibit different strain
specificity.
[0048] The antibiotics of use may belong to any class known to be
active against aiiy of the bacterial species known or thought
likely to be present in the biofilm. Preferably, the one or more
antibiotics will be administered after the one or more
bacteriophages such that bacteriophage replication has become
established before any antibiotic treatment begins. In this case,
antibiotic treatment may be delayed up to days from application of
the one or more bacteriophages, e.g. from 1 to 2, 3, 4, 5, 6, 7, 8,
9 or 10 days. Preferably, a sample will be taken from the infection
site to check that bacteriophage replication is occurring before
antibiotic treatment begins. Where a panel of bacteriophages is
employed with each member of the panel exhibiting different strain
specificity, it will suffice that at least a proportion of the
panel can target the bacterial infection. This may be a single
bacteriophage or more than one bacteriophage.
[0049] Where a panel of bacteriophages is employed, the
bacteriophages may be provided in the form of a single therapeutic
composition or as a number of separate compositions each comprising
one or more members of the panel. A suitable panel may consist of
two or more, three or more, four or more, five or more, or six or
more bacteriophage strains. Such a panel may comprise two, three,
four, five, six, seven, eight, nine, ten, fifteen, twenty or more
different bacteriophages. The bacteriophages may be from the same
or different taxonomical groups of viruses.
[0050] Bacteriophages with the potential to control bacterial
infections may be identified by a process of bioprospecting. This
involves the identification of such agents by assay of material
from sources rich in the target bacteria, and introduction of such
material to cultures of the target bacteria. A suitable sample may
be taken from sewage from a hospital, urban or other source.
[0051] Typically, sewage samples are mixed with powdered or liquid
bacterial growth media and with the target strains of bacteria
against which it is desired to isolate specific bacteriophages.
[0052] Samples are screened for the presence of suitable
bacteriophages by monitoring their effect on bacterial cells.
Typically this may involve determining bacterial death by observing
the formation of cleared zones in bacteria grown on solid
substrates ("plaques") or a loss of turbidity in liquid
culture.
[0053] Each of the bacteriophage strains selected for formation of
a panel for use in combined phage and antibiotic therapy as
discussed above will typically have activity against the same
target bacterial species. By activity is meant the ability of a
bacteriophage to infect that bacterial species and to have a
detrimental effect on the infected cells. This may be seen in the
death of some or all of the infected cells. Preferably the
bacteriophages will have activity against the target bacterial
species, but will have no activity or lower activity against other
bacterial species.
[0054] Once isolated, bacteriophages may be assayed against
multiple strains (isolates) of the target bacterial species in
order to determine their activity and specificity. These isolates
may be taken from patients either infected or colonised with a
bacterial species. Suitable isolates may also be obtained from
natural or environmental sources of the bacterial target strain,
such as soil samples. Methods of isolating bacteria from such
samples are well known in the art. For example, suitable P.
aeruginosa isolates for testing of bacteriophage panels may be
obtained from is known P. aeruginosa infections such as otitis
externa, topical infections, burn infections, nosocomial
infections, or other infections. Suitable isolates may also be
obtained from natural sources of P. aeruginosa, such as soil
samples.
[0055] As indicated above, it is particularly desirable to put
together a phage panel which exhibits a broader strain specificity
for the target bacterial species than any of the individual
selected phages. That is, the panel of bacteriophages is able to
kill a greater number of strains or isolates of the target
bacterial species than any of the individual selected
bacteriophages.
[0056] This may be achieved by including in the preparation a
number of bacteriophage strains each having different specificities
for the target bacterial isolates giving the preparation an overall
total effectiveness against many more strains than any of the
individual bacteriophages. The phage panel may include one or more
bacteriophage strains which are effective against a broad spectrum
of bacterial isolates of the target species so that the
bacteriophages in the preparation have overlapping effectiveness,
with some specific isolates being targeted by multiple
bacteriophages, thus helping to minimise any development of
resistance. Individual strains of the target bacterial species may
therefore be killed by one or more of bacteriophages making up a
preparation. The activity of bacteriophages against a range of
isolates, for example at least 50 isolates, may be tested and the
resulting information correlated to identify a group of at least
two different bacteriophages which have a combined effectiveness
against the target bacterial species that is greater than the
effectiveness of any of the individual bacteriophages. Such
development of a panel is exemplified by the development of a panel
of bacteriophages effective against Pseudomonas aeruginosa as shown
in FIG. 1.
[0057] Bacteriophages may be grown separately in strains (growth
strains) of the host (or a related species) that support growth to
high levels, titrated and combined at therapeutic concentrations.
Typically, this may range from 100 to 100,000,000 infectious units
per dose for each bacteriophage in the mixture.
[0058] The amount of each bacteriophage employed will depend upon
its virulence against the target bacterial species. Count bacterial
strains may be used in the development of a panel, i.e. bacterial
strains which are indicators for individual prospective members of
the panel. A count strain may permit at least 1000 times more
plaque formation by one prospective member of the phage panel than
any other. In this way, a panel that is consistently effective
against a wide range of bacterial isolates may be achieved.
[0059] As indicated above, combined phage/ antibiotic therapy
according to the invention may be particularly useful for example
in targeting bacterial infection comprising or consisting of
Pseudomonas aeruginosa. Such infection may be, for example, at the
site of a skin bum or other skin wound. It may be in the lung, an
ocular infection or an ear infection. In this context, such an
infection comprising P. aeruginosa will be understood to include an
infection consisting essentially of P. aeruginosa. Thus, phage
therapy according to the invention may be applied to an. infection
composed entirely, predominantly or significantly of P.
aeruginosa.
[0060] As previously noted above, the present invention provides
eight deposited bacteriophage strains that are shown herein to be
effective at killing a broad range of P. aeruginosa isolates, and
mutants thereof which retain the ability to target the same
bacterial species. The relevant bacteriophage strains which were
deposited at the National Collection of Industrial and Marine
Bacteria (23 St Machar Drive, Aberdeen, AB24 3RY, Scotland, UK) on
24 Jun. 2003 are as follows:
TABLE-US-00002 Reference NCIMB Deposit Number BC-BP-01 NCIMB 41174
BC-BP-02 NCIMB 41175 BC-BP-03 NCIMB 41176 BC-BP-04 NCIMB 41177
BC-BP-05 NCIMB 41178 BC-BP-06 NCIMB 41179 BC-BP-07 NCIMB 41180
BC-BP-08 NCIMB 41181
[0061] These bacteriophages may be employed therapeutically
individually or as part of a phage panel to combat P. aeruginosa
infection. For example, a phage panel for use in accordance with
the invention may comprise any two, three, four, five, six, or
seven or all eight of the deposited strains. Any of said deposited
strains may be substituted by a mutant thereof which exhibits
desired P. aeruginosa strain specificity. As indicated above, a
panel consisting of the six strains BC-BP-01, BC-BP-02, BC-BP-03,
BC-BP-04, BC-BP-05 and BC-BP-06 has been found to be particularly
favourable, for example, for veterinary use to target canine ear
infection either alone or more preferably in combination with
antibiotic therapy. However, utility of this phage panel is not
confined to such use. It may find use in targeting P. aeruginosa
infection in a variety of clinical situations.
[0062] In a further aspect, the invention provides a pharmaceutical
composition comprising one or more bacteriophages selected from the
eight deposited bacteriophage strains noted above and mutants
thereof which retain the ability to target P. aeruginosa, together
with a pharmaceutical carrier or diluent carrier. Suitable carriers
and diluents include isotonic saline solutions, for example
phosphate-buffered saline. The composition may be formulated for
parenteral, intramuscular, intravenous, subcutaneous, transdermal,
ocular or aural administration, e.g. a liquid formulation for
administration as eye or ear drops. Such a bacteriophage
preparation may be used directly, stored frozen in aqueous or other
solution with an appropriate cryoprotectant (e.g. 10% sucrose),
freeze dried and rehydrated prior to use, or rendered stable in
some other formulation including (but not limited to) tablet,
emulsion, ointment, or impregnated wound dressing or other
item.
[0063] In a still further aspect, there is provided a combined
product for simultaneous, separate or sequential administration of
a panel of bacteriophages to treat a bacterial infection comprising
or consisting of P. aeruginosa, each member of said panel having a
different strain specificity and wherein said panel consists of two
or more bacteriophages selected from NCIMB 41174, NCIMB 41175,
NCIMB 41176, NCIMB 411177, NCIMB 41178, NCIMB 41179, NCIMB 41180
and NCIMB 41181 and mutants thereof which retain the ability to
target P. aeruginosa. As indicated above, in an exemplified
embodiment, the panel consists of the six bacteriophages NCIMB
41174 to NCIMB 41179. These may be employed individually or more
preferably in a single pharmaceutical composition. A combined
product or composition comprising one or more of the above-noted
deposited bacteriophages may further comprise one or more
antibiotics for simultaneous, separate or sequential administration
to the one or more bacteriophages. Such a combined product or
composition may alternatively or additionally comprise an alginase.
The alginase may also be provided for simultaneous, separate or
sequential administration to the selected one or more
bacteriophages.
[0064] The target specificity of a bacteriophage may be altered by
the choice of substrate on which it is grown. That is, two
genetically identical bacteriophages may exhibit different target
specificity when they have been grown on different substrates. In
this case, a bacteriophage may be identified by the nucleotide
sequence of its genome. A bacteriophage having the same genomic
sequence as one of the eight deposited bacteriophage strains listed
above is considered to be the same bacteriophage, even if the
target specificity that it exhibits is not identical to that of the
deposited strain.
[0065] As noted above, the invention also extends to mutants of the
deposited strains which retain the ability to kill bacteria of the
target species. In particular, the invention extends to mutant
forms of these strains which retain similar or improved target
specificity as the strain from which they are derived. Thus, one or
more bacteriophages in a composition or combined product of the
invention may be mutants derived from these deposited strains which
retain the ability to infect and show activity against Pseudomonas
aeruginosa.
[0066] Suitable mutant bacteriophages may be derived from any one
of the eight deposited strains BC-BP-01, BC-BP-02, BC-BP-03,
BC-BP-04, BC-BP-05, BC-BP-06, BC-BP-07 and BC-BP-08. A suitable
mutant strain may retain the same target specificity as the strain
from which it is derived. That is, it may infect and kill the same
isolates or strains of the target bacterial species as the
deposited bacteriophage. Similarly, it may be ineffective against
the same bacterial isolates or strains as the deposited
bacteriophage. Alternatively, mutant bacteriophage strains may be
used. which have altered target specificity, being more or less
able to infect and kill particular isolates or strains of the
bacterial target species.
[0067] Suitable mutant bacteriophage strains may have a similar
genome to a deposited strain. That is, the nucleotide sequence of
the genome of a mutant bacteriophage may retain sequence identity
to the genome of the deposited bacteriophage from which it is
derived. Suitable mutant strains may retain at least 90%, at least
95%, at least 97%, at least 98% or at least 99% nucleotide sequence
identity to the genome of a deposited strain, across the whole
length of the genome. Alternatively, these levels of sequence
identity may be retained across only a part of the genome, for
example those parts of the genome required for target specificity.
In one embodiment, the genome of a mutant bacteriophage may
comprise a gene encoding a further therapeutic protein, as
explained below. In such a case, the bacteriophage genome may
consist of a genome having a degree of nucleotide sequence identity
as set out above, plus those sequences necessary for the expression
of the additional therapeutic protein.
[0068] The UWGCG Package provides the BESTFIT program which can be
used to calculate sequence identity (for example used on its
default settings) (Devereux et al (1984) Nucleic Acids Research 12,
387-395). The PILEUP and BLAST algorithms can alternatively be used
to calculate identity or line up sequences (typically on their
default settings), for example as described in Altschul S. F.
(1993) J Mol Evol 36:290-300; Altschul, S. F. et al (1990) J Mol
Biol 215:403-10. Identity may therefore be calculated using the
UWGCG package, using the BESTFIT program on its default settings.
Alternatively, sequence identity can be calculated using the PILEUP
or BLAST algorithms. BLAST may be used on its default settings.
[0069] Software for performing BLAST analyses is publicly available
through the National Centre for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pair (HSPs) by identifying short
words of length W in the query sequence that either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighbourhood word score threshold (Altschul et al, supra).
These initial neighbourhood word hits act as seeds for initiating
searches to find HSPs containing them. The word hits are extended
in both directions along each sequence for as far as the cumulative
alignment score can be increased. Extensions for the word hits in
each direction are halted when: the cumulative alignment score
falls off by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the accumulation of
one or more negative-scoring residue alignments; or the end of
either sequence is reached. The BLAST algorithm parameters W, T and
X determine the sensitivity and speed of the alignment. The BLAST
program uses as defaults a word length (W) of 11, the BLOSUM62
scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad.
Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of
10, M=5, N=4, and a comparison of both strands.
[0070] The BLAST algorithm performs a statistical analysis of the
similarity between two sequences; see e.g.. Karlin and Altschul
(1993) Proc. Natl. Acad. Sci. USA 90: 58735787. One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two polynucleotide or amino acid sequences
would occur by chance. For example, a sequence is considered
similar to another sequence if the smallest sum probability in
comparison of the first sequence to the second sequence is less
than about 1, preferably less than about 0.1, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0071] Mutations may he made to specific bacteriophages by
chemical, radiological or other methods well known to those skilled
in the art. Mutants with useful characteristics may then be
selected by assay of infectious, physical or genetic
characteristics, for example the ability to infect previously
resistant bacterial strains. Mutation may also be made by
homologous recombination methods well known to those skilled in the
art. The mutated sequence may comprise deletions, insertions or
substitutions, all of which may be constructed by routine
techniques. Insertions may include selectable marker genes, for
example lacZ, for screening recombinant viruses by, for example,
.beta.-galactosidase activity.
[0072] Insertions may also include sequences that encode proteins
desired for simultaneous administration with the bacteriophages, as
described in more detail below. For example, one or more of the
bacteriophages in a preparation of the invention may include a
sequence encoding an alginase such that the alginase is expressed
in an infected bacterial cell.
[0073] Bacteriophage-containing products of the invention can be
utilized in many situations where conventional antibiotic therapy
is problematic. In yet another aspect, the invention provides a
method of therapeutic or prophylactic treatment of a bacterial
infection characterised by biofilm formation which comprises
administering to a human or non-human animal in need thereof one or
more bacteriophages capable of targeting bacteria of said infection
and simultaneously, separately or sequentially thereto one or more
antibiotics. In one embodiment, the bacteriophages employed may be
one or more of the deposited bacteriophages noted above capable of
targeting P. aeruginosa infections. The invention also provides in
still further aspect a method of therapeutic or prophylactic
treatment of a bacterial infection comprising or consisting of P.
aeruginosa which comprises administering to a human or non-human
animal in need thereof one or more deposited bacteriophages or
mutants thereof as discussed above.
[0074] In particular, the preparations or strains of the invention
may be used to address chronic or antibiotic-resistant infections.
Thus, the use of bacteriophage preparations and strains as
specified may initially be as a secondary treatment where infection
is proving difficult to clear with existing antibiotics, or in
combination or rotation where there is a critical need for
clearance of infection. Thus, they may be used to complement and
supplement antibiotic use, especially, for example, in relation to
P. aeruginosa infections and other infections characterised by
biofilm formation
[0075] The treated infection may be in a human or animal, for
example a dog or a cat. The infection may be, for example, in or on
the ear, eye, skin or other topical location. The infection may be
systemic. Pseudomonas aeruginosa infections that may be treated by
the methods of the invention include otitis externa and other ear
infections, keratitis and other eye infections, folliculitis,
infections of bums and induced graft rejection, wound infections,
hospital acquired infections (nosocomial infections) and lung
infections, for example in cystic fibrosis. Urinary tract
infections and bacteraemias may also be treated.
[0076] Specific prophylactic uses of phage according to the
invention are: Ocular--inclusion in eye drops, contact lens
solutions or additives, embedded or otherwise included into contact
lenses, or otherwise formulated for administration into the eye for
prevention of P. aeruginosa infection.
[0077] Aural--inclusion in ear drops, ear plugs, impregnated
dressings (e.g cotton wool) devices for implantation (e,g. tympanic
membrane grafts, bone irsafts) or otherwise formulated for
administration into the ear for prevention of P. aeruginosa
infection. Wound dressings, salves, skin grafts and other
formulations for the prevention of the infection of body surfaces
or of devices within the body with P. aeruginosa. Formulation into
or for treatment of medical devices, e.g. artficial joints.
[0078] The preparations of the invention may further comprise, or
may be administered simultaneously, separately or sequentially with
further therapeutic agents. For example, treatment with a
preparation or strain of the invention may be coordinated with that
of another agent relating to the condition being treated, The
preparations or strains of the invention may be administered
alongside antibiotics to complement or supplement their actions.
The preparations or strains of the inventions may be administered
alongside agents directed to other aspects of the condition of the
patient, for example agents which may reduce inflammation,
stimulate or reduce an immune response, relieve pain or otherwise
improve the condition of the patient. The preparations or strains
of the invention may be administered alongside other agents being
used to treat a patient wherein the other agents may lead to an
increased risk of bacterial (e.g. Pseudomonas aeruginosa)
infection. For example, preparations or strains of the invention
may be administered to a patient suffering from immunosuppression,
such as localized immuncsuppression due to treatment with another
agent.
[0079] In one embodiment, the use of the preparations or strains of
the invention to treat a P. aeruginosa infection, for example a
lung infection, may be supplemented with the administration of an
alginase. As explained above, Pseudomonas has a tendency to grow in
a complex layer known as a biofilm. The biofilm is an assemblage of
surface-associated microbial cells that is enclosed in an
extracellular polymeric substance (EPS) matrix. Alginate is the
major component of the EPS matrix of P. aeruginosa. The use of
alginase may therefore help to disrupt such P. aeruginosa biofilms
and potentiate the clearance of infection. Biofilms may be present
in a variety of P. aeruginosa infections, including infections of
the lung and ear. Co-administration of an alginase with a
preparation or strain of the invention may be particularly suitable
for use in treating such conditions.
[0080] The alginase may be included in a composition of the
invention together with one or more bacteriophages or may be
provided in a separate composition for separate or sequential
administration to one or more bacteriophages. The alginase may be
provided from a sequence within the genome of a bacteriophage. This
may involve the isolation of such bacteriophages from environmental
sources or, for example, the genome of a bacteriophage may be
engineered by methods known in the art to include such a sequence
operably linked to suitable regulatory sequences.
[0081] The amount of bacteriophage administered will depend upon
the size, location and nature of the area to be treated and the
route of administration used. As a successful treatment will lead
to multiplication of the bacteriophages and killing of infected
bacteria, some treatments, for example those requiring topical
infection, may require only a low dose of bacteriophages. This dose
is measured in infectious units, usually defined by the ability to
form cleared zones or "plaques" on bacterial culture plates. Such
units are defined as "plaque forming units" or "pfu". For example,
in some cases the dose may be a few hundred infectious units (pfu)
or less. A suitable dose may be 10.sup.2 to 10.sup.8 pfu,
preferably 10.sup.4 to 10.sup.6 pfu. In other cases, for example in
a systemic or widespread infection, the dose may need to be higher
to ensure that the bacteriophages reach all infected areas. In such
a case a suitable dose may be in the range of from 10.sup.4 to
10.sup.10 pfu, preferably from 10.sup.5 to 10.sup.8 pfu. When
injected, typically 10 .mu.l to 1 ml of bacteriophages in a
pharmaceutically acceptable suitable carrier or diluent is
administered. For topical administration the volume may be higher,
for example 100 .mu.l to 50 ml of the medicament, depending on the
size, location and nature of the area to be treated.
[0082] Bacteriophage preparations and compositions of the invention
may be administered to the human or animal patient topically,
systemically, orally, or by some other means suitable for
delivering an effective dose to the site of the infection to be
treated. Bacteriophage administration will be in such a way that
the bacteriophage can be incorporated into bacteria at the site of
infection. The routes of administration and dosages described are
intended only as a guide since a skilled practitioner will be able
to determine the optimum route of administration and dosage for any
particular patient and condition.
[0083] As indicated above, the invention also extends to
non-therapeutic methods of removing, reducing or preventing
bacterial contamination characterised by biofilm formation. This in
yet another aspect, the invention provides such a method comprising
applying to the site or prospective site of such contamination one
or more bacteriophages capable of targeting appropriate bacteria
and simultaneously, separately or sequentially thereto one or more
antibiotics or antiseptics. Such a method may, for example, be
applied to P. aeruginosa contamination in which case one or more of
the above-noted deposited bacteriophages may again be employed or a
mutant thereof. One or more of the same bacteriophages may also be
employed non-therapeutically alone to target bacterial
contamination comprising or consisting of P. aeruginosa. Such
methods may be applied for the treatment of a variety surfaces in
both medical and non-medical contexts, e.g. contact lenses,
surfaces of devices to be implanted into the body, pipes, ducts and
other surfaces where bacterial infections can become
established.
[0084] Preparations and strains of the invention may also he used
in in vitro diagnostic methods to detect the presence of
Pseudomonas aeruginosa. Such methods may comprise the steps of
contacting a test sample with one or bacteriophages capable of
targeting P. aeruginosa as discussed above and determining whether
any of the bacteriophages thus added are capable of killing
bacteria in the test sample. Preferably, the test sample will be
cultured prior to contact with the preparation or strain, for
example under conditions suitable to allow growth of any bacteria
of the target species that are present. Suitable culture conditions
are known in the art, and will depend upon the specific bacterial
target species.
[0085] A panel of bacteriophages of the invention capable of
targeting P. aeruginosa is particularly useful for such methods
because it will detect a broad spectrum of bacterial strains or
isolates. A single bacteriophage from a preparation of the
invention may detect only a small proportion of strains or isolates
of a particular bacterial species, and will therefore typically
offer a very high false negative rate simply because of this high
specificity. However, the use of a preparation of the invention,
comprising two or more such bacteriophages, will allow detection of
a broad spectrum of strains within a target bacterial species.
[0086] In one embodiment, the test sample may be cultured on a
solid growth medium, such as on an agar plate. The sample is
preferably cultured on said medium for a sufficient time and tinder
suitable conditions for any target bacteria present in the sample
to multiply on the surface of the plate. By contacting the surface
of the plate with a preparation or strain of the invention, it can
be determined whether any of the bacteriophages thus added are
capable of infecting and killing the bacteria. The
bacteriophage-infected medium may be maintained under suitable
conditions for bacteriophage infection and replication, such that
the bacteriophages have an opportunity to infect any target P.
aeruginosa cells on the plate. This will lead to the development of
clear patches (plaques) where bacterial death has occurred, and
will indicate that the test sample contained the target bacterial
species.
[0087] In an alternative embodiment, the test sample may be
maintained in a liquid median. Again, it may be cultured under
conditions suitable for bacterial growth. Following the addition of
a preparation or strain of the invention, the medium may be
maintained for a further period to allow the bacteriophages to
infect any target bacteria present. This will lead to a loss of
turbidity in the medium when bacterial death occurs, and this will
indicate that the test sample contained the target bacterial
species.
[0088] The test sample may be any sample where the presence of the
target bacterial species is suspected. The test sample may be from
an environmental or biological source. Such a test sample may be
from, or derived from, a fluid or tissue sample obtained from the
patient. The sample may be obtained from the location of an
infection. In the case of a topical infection, the sample may be
obtained by taking a swab from the infected region. The detection
method of the invention may be used to determine particular strains
of P. aeruginosa responsible for the infection.
[0089] An infection capable of being identified using a preparation
or strain of the invention will normally be treatable using the
same preparation or strain. That is, if the bacteriophages in a
preparation of the invention are capable of killing bacteria
obtained from the infected area in vitro, they should also be
capable of killing the same bacteria in situ at the site of
infection.
[0090] The detection method of the invention may therefore also be
used to identify a suitable preparation or strain of the invention
for use in treatment. The detection method of the invention may
also be used to identify single bacteriophages that are suitable
for use in such treatment individually, rather than in combination.
That is, by using different bacteriophages or combinations of
bacteriophages in the detection methods of the invention, the
bacteriophage(s) with the greatest virulence towards the bacterial
strain of the specific infection may be selected for use in
treatment. Preferably, a combination of two or more bacteriophages
having such activity is selected for use in treatment.
[0091] The present invention also includes the identification and
use of bacterial "count strains" for the deposited bacteriophages
noted above. Such bacterial strains are defined as strains of the
target (or a related) bacterium which support the growth of one
bacteriophage from the specified group while only permitting
limited growth of all other bacteriophage components of the group.
These count strains may then be used to assess titres of the
bacteriophage stocks.
[0092] The preparations of the invention comprise at least two
bacteriophages. Across a spectrum of bacterial strains, the growth
of any one bacteriophage will be supported with varying efficiency
(or not at all). Consequently, titres obtained by assaying across a
range of bacterial strains will differ substantially. In order to
so provide a means of determining/standardising the therapeutic
dose to be administered to each patient, `count` bacterial strains
may be used. The principle of this approach is that the growth of
each bacteriophage is only supported at a usable level by one of
the range of bacterial strains. A count strain can thus be selected
for each of the bacteriophages in a mixture which supports the
growth of one of the bacteriophages but does not support the
growth, or only supports a low level of growth, of the other
bacteriophages in the mixture. Titres of each bacteriophage
constituent of the mixture may be calculated based on the growth
possible on each of the individual count strains.
[0093] For example, a suitable count strain may allow at least 1000
times more, at least 1500 times more, at least 2000 times more
growth, or greater, of one bacteriophage compared to the other
bacteriophages being used in a mixture of bacteriophages.
[0094] The differential growth may be assessed, for example by
titrating plaque formation on the bacteria when grown on solid
arowth medium by the bacteriophages at a range of concentrations.
Alternatively, the differential growth may be assessed by looking
at the size and nature of the plaques so formed. For example, in
one embodiment, the count strain may allow at least 1000 times more
plaque formation by one bacteriophage than other bacteriophages.
Such a bacterium would form a count strain for that bacteriophage.
For example, as shown in FIG. 2, a suitable growth strain for
BC-BP-03 may show significant plaque formation following infection
with BC-BP-03 at a 1,000,000-fold dilution, but little or no plaque
formation when infected with other bacteriophages (here BC-BP-01,
BC-BP-02 and BC-BP-04) at a 10-fold dilution (100,000 times higher
concentration).
[0095] Such a count strain may also be used as a propagation strain
for production of bacteriophage for use in the compositions of the
invention. For example, a composition of the invention may be
formed by combining the required count strains in the appropriate
quantities. The count strains may therefore have a therapeutic use
themselves as a source of bacteriophages.
[0096] This technique also enables the replication of each
bacteriophage in a therapeutic composition to be monitored
individually in a clinical context. Count strains specific for a
particular bacteriophage may be used to identify the presence of
that bacteriophage in particular, for example in preparations
intended for therapeutic use or in tissue samples obtained during
or following such therapeutic use. It is anticipated that this
method will allow therapeutic bacteriophages to be distinguished
from any extraneous bacteriophages that might exist in the strain
with which the patient is infected, and would also allow the
determination of which bacteriophages in the administered
therapeutic mixture are active against that patient's bacterial
infection. The `count` bacterial strains can be used to `type` any
extraneous bacteriophage prior to administration of the
bacteriophage therapy. The presence of the required bacteriophages
in a composition or medicament may therefore be confirmed prior to
treatment and the presence of the bacteriophages at the treatment
site may be monitored during and after treatment. This information
may be used by the medical practitioner to monitor and adjust the
treatment regimen.
The following examples illustrate the invention.
EXAMPLES
[0097] I. Initial Selection of Bacteriophages Active Against
Pseudomonas aeruginosa (a) Isolation of Bacteriophages Active
Against Pseudomonas aeruginosa:
[0098] (i) 3.times.10.sup.9 colony forming units (cfu) of
appropriate Pseudomonas aeruginosa strain cultured with settled
sewage and nutrient broth (total volume 200 ml).
[0099] (ii) Suspension incubated at 37.degree. C. for 24 hours.
[0100] (iii) 1 ml aliquot removed and filtered through 0.45 .mu.m
syringe-top filter.
[0101] (iv) Filtered lysate cultured with the same Pseudomonas
aeruginosa strain used in step (i), and assessed for presence of
bacteriophage (see below)
[0102] (v) Nutrient agar plates incubated at 37.degree. C. for 24
hours.
[0103] (vi) Single plaque `picked` using sterile 1 mm diameter wire
and used to inoculate 3 ml of growth media (the constituents of
this media varied between extractions) containing 5.times.10.sup.6
cfu/ml of the Pseudomonas aeruginosa strain used in step (i).
[0104] (vii) Suspension incubated at 37.degree. C. until lysis of
bacteria complete (this typically takes between 5-8 hours) and is
assessed visually. The visual assessment is facilitated by
comparing the turbidity of the bacteriophage-containing bacterial
suspension with that of a control suspension. Control suspensions
do not receive bacteriophage, yet are similar in every other
respect.
[0105] (viii) Lysate filtered through 0.1 .mu.m syringe-top
filter.
[0106] (xi) Lysate adjusted to constitute 2% v/v glycerol,
aliquotted into vials and stored at -80.degree. C.
[0107] (xii) Titres assessed by co-culturing with appropriate
bacterial strain (see below)
(b) Preparation of Master Seeds:
[0108] Master seed stocks are established for all bacteriophages as
follows:
[0109] (i) Primary bacteriophage preparations were co-cultured with
appropriate Pseudomonas aeruginosa propagating strain on agar
plates (see below)
[0110] (ii) Single plaques `picked` using sterile 1mm diameter wire
and used to inoculate 4 ml of Vegetable Peptone Broth containing
5.times.10.sup.6 colony forming units (cfu)/ml of the Pseudomonas
aeruginosa strain used in step (i).
[0111] (iii) Suspension incubated at 37.degree. C. until lysis of
bacteria complete (this typically takes between 5-8 hours) and is
assessed visually. The visual assessment is facilitated by
comparing the turbidity of the bacteriophage-containing bacterial
suspension with that of a control suspension. Control suspensions
do not receive bacteriophage, but are similar in every other
respect.
[0112] (iv) Lysate filtered through 0.1 .mu.m syringe-top
filter.
[0113] (v) Master stock adjusted to constitute 2% v/v glycerol,
aliquotted into vials and stored at -80.degree. C.
[0114] (vi) Titres assessed by co-culturing with appropriate
bacterial strain(see below)
(c) Assessment of Titres of Individual Bacteriophages Within a
Mixed Population: Bacteriophage vs Pseudomonas aeruginosa `Count`
Bacterial Strains
[0115] (i) Each bacteriophage (individual suspension) was assayed
in duplicate on all count strains. The Master seeds were used.
[0116] (ii) Titres were calculated for each bacteriophage on each
`count` bacterial strain.
[0117] (iii) Steps (i)-to-(ii) were repeated on two further
occasions.
[0118] (iv) On the final occasion the mixed bacteriophage
suspension containing equal proportions of the 6 individual
bacteriophage preparations was assayed in duplicate on all `count`
bacterial strains.
[0119] (v) Titres for bacteriophage mix on each `count` bacterial
strain were calculated
[0120] All three sets of experiments yielded comparable results,
which are detailed in Table 1, along with an averaged result
TABLE-US-00003 TABLE 1a Experiment #1--Bacteriophage vs `count`
bacterial strains Titre (pfu/ml) (Titres presented are means of
duplicate readings) P. aeruginosa strain Count Count Count Count
Count Count Bacteriophage 06 02 03 01 04 05 BC-BP-06 -- -- -- -- --
BC-BP-04 -- -- Barely -- -- discernible inhibition. No plaques
BC-BP-02 -- Barely -- -- -- discernible inhibition. No plaques
BC-BP-05 -- -- -- -- -- BC-BP-01 -- -- 6 .times. 10.sup.2 --
`Turbulences` observed at 10.sup.-1 and 10.sup.-2 Dilutes out
BC-BP-03 -- -- 2.2 .times. 10.sup.3 -- --
TABLE-US-00004 TABLE 1b Bacteriophage vs cross-reacting `count`
bacterial strains; difference in titres (experiment #1) Titre
(pfu/ml) (Titres presented are means of duplicate readings) P.
aeruginosa strain Bacteriophage Count 03 Count 01 Fold difference
BC-BP-01 .sup. 6 .times. 10.sup.2 2.84 .times. 10.sup.8 4.73
.times. 10.sup.5 BC-BP-03 3.1 .times. 10.sup.8 2.2 .times. 10.sup.3
1.4 .times. 10.sup.5
TABLE-US-00005 TABLE 1c Experiment # 2--Bacteriophage vs `count`
bacterial strains Titre (pfu/ml) (Titres presented are means of
duplicate readings) P. aeruginosa strain Count Count Count Count
Count Count Bacteriophage 06 02 03 01 04 05 BC-BP-06 -- -- -- -- --
BC-BP-04 -- -- Barely -- -- discernible inhibition. No plaques
BC-BP-02 -- Barely -- -- -- discernible inhibition. No plaques
BC-BP-05 -- -- -- -- -- BC-BP-01 -- -- 3.5 .times. 10.sup.2 --
`Turbulences` (poor observed at duplicates) 10.sup.-1 and 10.sup.-2
Dilutes out BC-BP-03 -- -- 7 .times. 10.sup.2 -- --
TABLE-US-00006 TABLE 1d Bacteriophage vs cross-reacting `count`
bacterial strains; difference in titres (experiment #2) Titre
(pfu/ml) (Titres presented are means of duplicate readings) P.
aeruginosa strain Bacteriophage Count 03 Count 01 Fold difference
BC-BP-01 3.5 .times. 10.sup.2 8.1 .times. 10.sup.7 2.3 .times.
10.sup.5 BC-BP-03 2.65 .times. 10.sup.8 .sup. 7 .times. 10.sup.2
3.8 .times. 10.sup.5
TABLE-US-00007 TABLE 1e Experiment #3--Bacteriophage vs `count`
bacterial strains; individual and mixed. Titre (pfu/ml) (Titres
presented are means of duplicate readings) Outcome of experiments
where the 6 bacteriophages are mixed before assaying shown in bold
text P. aeruginosa strain Count Count Count Count Count Count
Bacteriophage 06 02 03 01 04 05 BC-BP-06 -- -- -- -- -- 5.8 .times.
10.sup.9 BC-BP-04 -- -- Barely -- -- discernible 9.6 .times.
10.sup.8 inhibition. No plaques BC-BP-02 -- Barely -- -- -- 7.5
.times. 10.sup.8 discernible inhibition. No plaques BC-BP-05 -- --
-- -- -- 1.4 .times. 10.sup.9 BC-BP-01 -- -- 1 .times. 10.sup.2 --
`Turbulences` 3.3 .times. 10.sup.8 observed at 10.sup.-1 and
10.sup.-2 Dilutes out BC-BP-03 -- -- 7.5 .times. 10.sup.2 -- --
3.35 .times. 10.sup.8
TABLE-US-00008 TABLE 1f Bacteriophage vs cross-reacting `count`
bacterial strains; difference in titres (experiment #3) Titre
(pfu/ml) (Titres presented are means of duplicate readings) P.
aeruginosa Strain Bacteriophage Count 03 Count 01 Fold difference
BC-BP-01 1 .times. 10.sup.2 3.3 .times. 10.sup.8 3.3 .times.
10.sup.6 BC-BP-03 3.35 .times. 10.sup.8 7.5 .times. 10.sup.2 4.5
.times. 10.sup.5
TABLE-US-00009 TABLE 1g Bacteriophage vs `count` bacterial strains;
mean results of experiment #1, #2 and #3 Titre (pfu/ml) (Titres
presented are means of duplicate readings in experiments #1, #2 and
#3) P. aeruginosa strain Count Count Count Count Count Count
Bacteriophage 06 02 03 01 04 05 BC-BP-06 -- -- -- -- -- BC-BP-04 --
-- Barely -- -- discernible inhibition. No plaques BC-BP-02 --
Barely -- -- -- discernible inhibition. No plaques BC-BP-05 -- --
-- -- -- BC-BP-01 -- -- 3.5 .times. 10.sup.2 -- `Turbulences`
observed at 10.sup.-1 and 10.sup.-2 Dilutes out BC-BP-03 -- -- 1.2
.times. 10.sup.3 -- --
TABLE-US-00010 TABLE 1h Bacteriophage vs cross-reacting `count`
bacterial strains; difference in titres. Means of experiments #1,
#2 and #3 Titre (pfu/ml) (Titres presented are means of duplicate
readings in experiments #1, #2 and #3) P. aeruginosa strain
Bacteriophage Count 03 Count 01 Fold difference BC-BP-01 3.5
.times. 10.sup.2 2.3 .times. 10.sup.8 6.6 .times. 10.sup.5 BC-BP-03
.sup. 3 .times. 10.sup.8 1.2 .times. 10.sup.3 2.5 .times.
10.sup.5
(d) Preparation of Purified Bacteriophage Suspensions:
[0121] Bacteriophages are prepared for use from master suspensions
as follows:
[0122] 30 ml suspensions of appropriate growth strains of
Pseudomonas aeruginosa in Vegetable Peptone Broth inoculated with
master seeds of the appropriate bacteriophage at a multiplicity of
infection of 0.1.
[0123] (ii) Suspension incubated at 37.degree. C. until lysis of
bacteria complete (this typically takes between 5-8 hours) and is
assessed visually. The visual assessment is facilitated by
comparing the turbidity of the bacteriophage-containing bacterial
suspension with that of a control suspension. Control suspensions
do not receive bacteriophage, but are similar in every other
respect.
[0124] (iii) Sub-master seeds filtered through 0.45 .mu.m then 0.1
.mu.m syringe-top filters.
[0125] (iv) 27 ml of the sub-master seeds carefully over-layed onto
5 ml of a 10% w/v sucrose `cushion`, in 36 ml polypropylene
centrifuge tubes. The purpose of the sucrose `cushion` is to
prevent the sedimentation of endotoxins, while allowing the virus
particles to pellet at the bottom of the tube.
[0126] (v) All centrifuge tubes and buckets thoroughly cleaned and
then autoclaved at 121.degree. C. prior to use.
[0127] (vi) Tubes spun at 23,500 rpm at 4.degree. C. for 3 hours in
a Beckman ultra-centrifuge
[0128] (vii) Supernatant fractions removed and the pellets drained.
Pellets then re-suspended in 1 ml PBS+10% v/v glycerol and filtered
through 0.2 .mu.m syringe-top filters.
[0129] (vi) Titres assessed by co-culturing with appropriate
bacterial strain (see below)
[0130] This material can be used in vivo, subject to sterility
controls:
(e) Sterility
[0131] The final product was tested for sterility as follows:
[0132] (i) Three 0.6 ml aliquots of final therapeutic product
randomly selected
[0133] (ii) Each aliquot spread out on nutrient agar plate
(permissive for growth of a range of bacterial species including
Pseudomonas) using sterile wire loop
[0134] (iii) Plates incubated at 37.degree. C. for 48 hours
[0135] (iv) Plates checked for presence of bacterial colonies
[0136] (Such tests on the material as prepared showed no bacterial
growth)
(f) Assessment of Efficacy:
[0137] For the bacteriophage product designed to combat Pseudomonas
aeruginosa strains which cause canine otitis externa, the
development of the product entailed selection of appropriate
bacteriophages to fulfil this role. This was achieved by
co-culturing a range of 22 bacteriophages with 100 clinical
Pseudomonas aeruginosa isolates derived from canine otitis externa
infections as indicated below. It was found that 90% of these
strains were susceptible to at least one of the 6 of the candidate
bacteriophages BC-BP-01 to BC-BP-06 (FIG. 1) from the initial panel
of 22 strains. These 6 bacteriophages were progressed into clinical
trials on the basis that this in vitro data supported the
expectation that the product will be clinically effective in
vivo.
Method
[0138] (i) Bacteriophage preparation diluted in. PBS in a 10-fold
dilution series at room temperature.
[0139] (ii) 100 .mu.l of appropriate dilution(s) added to 2.5 ml
molten agar at 46.degree. C. containing 5.times.10.sup.6cfu of
relevant bacteria.
[0140] (iii) Molten agar suspensions poured onto nutrient agar
plates and allowed to set at room temperature.
[0141] (iv) Plates transferred to 37.degree. C. and incubated for
24 hours.
[0142] (v) Plaques counted and numbers used to calculate titre in
pfu/ml. One is aiming to count the dilution which gives around 100
plaques per plate.
(g) Assessment of Safety:
[0143] A veterinary clinical trial was conducted to assess the
toxicity of the bacteriophage product. The total duration of the
study was 21 days. Six dogs (3 male; 3 female) received the
following treatment regimen administered aurally on 3 occasions, at
days 0, 3 and 6 of the study:
TABLE-US-00011 Number Dose Dose of Volume Volume Group animals Sex
Treatment Left Ear Right Ear 1 2 1 1 Diluent 0.2 ml 0.2 ml
(control) Male Female 2 4 2 2 Bacterio- 0.2 ml 0.2 ml (test) Male
Female phage (10.times. (100.times. therapeutic therapeutic dose)
dose)
[0144] Administration of the treatment was made by drops of liquid
suspension into the external ear canal which was then massaged to
promote deep penetration.
[0145] The following investigations were undertaken during the
study:
[0146] (i) Microbiological flora at days 0, 3 and 6 (samples taken
immediately prior to administration of treatment) were assessed by
plating of ear swabs on: [0147] 1) Cetrimide agar, selective for
Pseudomonas spp [0148] 2) Mannitol salt agar, selective for
Staphylococcus spp [0149] 3) Sabouraud dextrose agar, selective for
yeast and moulds [0150] 4) FP agar, selective for micrococci [0151]
5) Blood agar, nonselective permitting growth of most
microorganisms
[0152] (ii) Auroscopic veterinary examination daily until day 8 of
the study, then every three days until the conclusion of the
study
[0153] (iii) Core temperature measured daily
[0154] Throughout the study:
[0155] (i) Auroscopic veterinary examination revealed no
significant changes in the condition of the ears of dogs in the
test group, as compared with baseline recordings and the control
group.
[0156] (ii) There were no significant changes in the
microbiological flora within the ears of test group dogs, as
compared with baseline recordings and the control group.
[0157] (iii) In the test group, core temperature recordings did not
differ significantly from those noted in the control group or at
baseline.
II. Assessment of Clinical Efficacy of Selected Bacteriophages
[0158] (a) Protection of Mice from Lethal Infection with
Pseudomonas aeruginosa
[0159] 1. 150,000,000 infectious units (10 LD.sub.50) of
Pseudomonas aeruginosa were injected into the peritoneal cavities
of 20 mice
[0160] 2. Groups of 5 mice were treated with 4 different
concentrations of bacteriophage BC-BP-08, administered
simultaneously with the bacterial injection.
TABLE-US-00012 TABLE 2 Mouse survival after 150,000,000 infectious
units (10 LD.sub.50) of Pseudomonas aeruginosa were injected into
the peritoneal cavities of 20 mice Bacteriophage (infectious units)
Not surviving Surviving 290,000,000 0 5 29,000,000 4 1 5,800,000 5
0 290,000 5 0
(b) Prevention of the Destruction of Pig Skin in vitro by
Pseudomonas aeruginosa
[0161] Fourteen wound models were made each consisting of four
layers of enzyme cleaned, sterilized, freeze dried pig dermis
soaked in human plasma. 100,000 cfu of P. aeruginosa were placed on
the top of each. Seven of the wound models received 1,000,000
infectious units of phage BC-BP-08, the remainder serving as
controls. After 18 hours incubation, 7 of 8 of the controls were
blindly assessed as decayed, whereas all of the phage treated
models were assessed as not decayed. Bacteria could only be
detected in three of the treated discs (median count 0; highest
value 12,000). Phages were found to have penetrated through to the
bottom layer of the model and have replicated in it (median count
in models after incubation: 32,000,000; range
14,000,000-20,000,000,000).
(c) Protection of Skin Grafts on Guinea Pigs from Infection with
Pseudomonas aeruginosa
[0162] 1. A 0.2mm thick rectangle of shaved skin (2cm.times.1 cm)
was excised from the backs of 14 guinea pigs
[0163] 2. The underlying skin layers were removed to make the
wounds comparable to an excised burn
[0164] 3. 600,000 infectious units of Pseudomonas aeruginosa were
introduced into the wounds
[0165] 4. 12,000,000 infectious units of the bacteriophage BC-BP-08
were introduced into the wounds on 7 animals, with no bacteriophage
introduced into the wounds of the other 7
[0166] 5. The skin rectangle was replaced and dressed
[0167] 6. Grail success was assessed after 5 days
TABLE-US-00013 TABLE 3 Protection of skin grafts on guinea pigs
from infection with Pseudomonas aeruginosa Graft succeeded Graft
failed No bacteriophage 0 7 With bacteriophage 6 1
(d) Multiplication of Administered Pseudomonas aeruginosa
bacteriophages in the External Auditory Canal of a Dog with otitis
externa
[0168] The dog, with a history of atopy, had bilateral chronic
otitis externa. Swabs of both ears had repeatedly grown P.
aeruginosa for many months, despite antibiotic therapy. Before
phage therapy, the dog had bilateral swollen erythematous external
auditory canals, each with a purulent discharge and copious waxy
secretions over the surrounding pinna. Swabs taken from each ear at
that time grew as P. aeruginosa (identified by API 20NE,
Biomerieux, France). The isolates from the two ears had differed
slightly in their biochemical reactions and in their antibiotic
sensitivity patterns. From a phage collection, eight were selected
for testing against the strains, because they had previously been
identified as exhibiting good lytic activity against a wide range
of P. aeruginosa strains. Three of the phages displayed good lytic
activity against both isolates obtained from the dog. The phages
were further tested for how few plaque-forming units would lyse a
standard broth culture of the two isolates. BC-BP-04, the phage for
which the number of pfu required for lysis was lowest was chosen
for in vivo work. The phage concentration in the stock solution.
had been titrated, with 0.2 ml of a 10.sup.5 dilution of this
solution being applied by syringe to the dog's right external
auditory meatus. This volume contained approximately 400 infectious
units (plaque forming units, p.f.u.).
[0169] Twenty seven hours after phage application, a swab was taken
of the detritus within the right ear. It was weighed before and
after swabbing and the mass of detritus was determined. Phages were
counted in the detritus by plating serial 10 fold dilutions using
the agar overlay technique. There were 1.6.times.10.sup.8 phages in
the 0.032 g of detritus that was present on the swab, so it is
likely that the phage multiplied over 1 million fold. This increase
was accompanied by a marked clinical improvement of the right ear.
There was less inflammation; disappearance of the purulent
discharge; and reduced amount of waxy secretions (FIG. 3a). The
appearance of the left ear, which had not received phage, remained
unchanged (FIG. 3b). In view of the change, 400 pfu of the phage
was applied to the left ear, which was followed by considerable
clinical improvement 24-48 hours later. Two weeks after the phage
application, the dog's ears had deteriorated; and swabs of both
were positive for P. aeruginosa on culture. Subsequently the
condition of the ears repeatedly deteriorated and improved for many
months but the owner and veterinarian judged that their condition
was better than it had been before phage was administered. Nine
months after phage administration both ears completely recovered,
and P. aeruginosa has not since been isolated from aural swabs. No
antibiotics were given to the dog after phage administration as
they were not considered necessary.
(e) Use of a Pseudomonas aeruginosa bacteriophage in the Treatment
of an Infection of a Human Burn
[0170] A single case trial was carried out on a 27 year old man
with 50% burns. Episodes of healing of the burns alternated with
periods of breakdown of the skin. It was noted that the skin on his
back and chest were breaking down. At this time the clinicians, who
were concerned by the rapid rate of breakdown of the skin, asked if
a phage could be found to treat his P. aeruginosa infection. A new
phage, BC-BP-07, was isolated that was active. Although in vitro
testing did not indicate great activity against the strain, time
was limited, so it was selected for further work. A purified
suspension was made and no evidence of toxicity of which was shown
when it was added to cultures of human epidermal cells.
Approximately 1000 infectious units (p.f.u.) of BC-BP-07 were
applied to each of two filter paper discs of diameter 25 mm. At a
dressing change these were placed on areas of the patient known to
be colonised with P. aeruginosa. 48 hours later counts of phage in
the discs were 1.2.times.10.sup.6 and 4.3.times.10.sup.4, increases
of 1,200.times. and 43.times.. After this the patient's burns were
sprayed with the phage. Following this, the patient's condition
gradually improved and he survived and eventually all wounds healed
over. Whether the phage contributed to the recovery of the patient,
who was also given antibiotics, is unknown, but phage did multiply
on the burns and demonstrates the bacteriophage multiplying in or
on a patient, thus indicating killing of bacteria by the
bacteriophage.
[0171] Prior to their deposit at NCIMB on 24 Jun. 2003, none of the
bacteriophages referred to herein as BC-BP-01 to BC-BP-08 were
publicly available, hence any reference to such strains in any
publication or other disclosure before that date does not represent
enabling prior art.
[0172] In the further exemplification provided below, use was made
of a combination of the six bacteriophages NCIMB 41174, NCIMB
41175, NCIMB 41176, NCIMB 41177, NCIMB 41178 and NCIMB 41179 (the
BioVet-PA composition) which had been found to be active against
90% of the Pseudomonas aeruginosa isolates tested from canine ear
infections (otitis externa and other ear infections). 0.2 ml of
BioVet-PA contained 1.times.10.sup.5 infectious units of each of
the six bacteriophages as measured against appropriate count
strains as described above.
[0173] The six individual purified bacteriophage suspensions were
diluted in 10% v/v glycerol:PBS to a concentration of approximately
3.times.10.sup.6 pfu/ml. This dilution step was based on titres
calculated from the samples of the bacteriophage suspensions that
had been frozen at -80 .degree. C. , then thawed and assayed. After
dilution, the six therapeutic bacteriophages were mixed together in
equal proportions, thus diluting each bacteriophage by a factor of
six and bringing the concentration of the individual constituents
to 5.times.10.sup.5pfu/ml. This is equivalent to 1.times.10.sup.5
pfu of each therapeutic bacteriophage in 0.2 ml diluent. At this
point, the final mixed product was aliquoted into 0.6 ml aliquots
and stored at -80 .degree. C.
III. Trial of a Combined Phage Composition Against Canine Ear
Infections
[0174] As indicated above, canine ear infections caused by
Pseudomonas aeruginosa (otitis externa and otitis media) are
examples of clinical disease associated with biofilm-based
colonization of a body surface. Clinical signs of such infection
include pain, irritation (erythema), ulceration and the discharge
of increased amounts of material from the ear. This is often
purulent in nature and is accompanied by a distinctive odour. The
BioVet-PA combined preparation of six bacteriophages noted above
was authorized for trial in dogs with such infection by the
Veterinary Medicines Directorate of the United Kingdom under Animal
Trials Certificate 20505/0001 issued to Biocontrol Limited on 17th
Nov. 2003.
Conduct of the Trial
[0175] BioVet-PA was stored at -80.degree. C. Immediately prior to
administration, the product was thawed and warmed in the hand. 0.2
ml (containing 1.times.10.sup.5 infectious units of each of the 6
bacteriophages) was administered drop-wise using a sterile 1 ml
capacity syringe into the ear. Ear condition and microbiology was
assessed at 2 days post-administration.
[0176] The procedure was as follows:
[0177] Characterisation (2 to 14 days prior to treatment):
TABLE-US-00014 Day 0 Swabs taken from each ear by a veterinary
surgeon Laboratory tests carried out using these swabs to confirm
presence of P. aeruginosa.
[0178] If P. aeruginosa was not detected, the dog was excluded from
the trial
TABLE-US-00015 Day 1 If P. aeruginosa was detected, the isolates
were tested for sensitivity to BioVet-PA,
[0179] If the P. aeruginosa strain(s) with which the dog was
infected was not sensitive to BioVet-PA, the dog was excluded from
the trial.
[0180] Treatment:
TABLE-US-00016 Day 0 Ears examined auroscopically to assess their
condition. Swabs taken from each ear for microbiological analysis,
Dog's core temperature measured Dog given dose of 0.2 ml BioVet-PA
into the ear (treatments administered drop-wise using a sterile 1
ml-capacity syringe, and ear canals then massaged to promote deep
penetration). Day 1 Ears examined to assess their condition. Swabs
taken from each ear for microbiological analysis. Dog's core
temperature measured. Day 2 Ears examined to assess their
condition. Swabs taken from each ear for microbiological analysis.
Dog's core temperature measured.
Results:
[0181] Studies on six dogs with severe, antibiotic-resistant
Pseudomonas aeruginosa ear infections treated with BioVet-PA showed
improvement in clinical symptoms within two days of treatment (FIG.
4) and reductions in bacterial numbers over the same timescale
(FIG. 5). Bacteriophage replication was also observed in all dogs
(FIG. 6). Analysis of the improvement in clinical symptoms showed
this to be significant at the 95% level of confidence by both the
t-test and the Wilcoxon matched-pairs test.
IV. Trial of a Combined Phage Composition with Antibiotics Against
Canine Ear Infections
[0182] The BioVet-PA composition plus antibiotics were used against
Pseudomonas aeruginosa ear infections in dogs that had proved
refractory to antibiotic treatment alone.
Case 1
[0183] Dog M had a history of bilateral otitis, which had failed to
resolve in the right ear despite repeated courses of treatment with
antibiotics, including marbofloxacin and gentamicin, which are used
to treat Pseudomonas otitis. At the start of the phage trial
(26/01/04), the right ear was infected with both Pseudomonas
aeruginosa and a group G beta-haemolytic Streptococcus. Examination
showed erythema, ulceration, and pu.n.ilent discharge accompanied
by odour. Following examination, dog M was treated with BioVet-PA
(100,000 infections units of each of the six bacteriophages in 0.2
ml of diluent).
[0184] Analysis following treatment showed that five of the six
bacteriophages were replicating. This was accompanied by a fall in
the number of Pseudomonas bacteria present in the ear and by
improvement in clinical symptoms. Eight days after the completion
of a two day monitoring period following bacteriophage treatment,
dog M was treated with Synulox (amoxicillin and clavanulate) for
the accompanying Streptococcus infection. This therapy also has
some activity against some Pseudomonas strains.
Result
[0185] Analysis of a swab taken on Mar. 4, 2008 showed no
detectable Pseudomonas, accompanied by low levels of Streptococcus.
This demonstrated the efficacy of bacteriophages in resolving
bacterial infection in a system characterised by biofiltn formation
when antibiotics and other chemical agents had previously failed to
clear the infection, along with improved results when antibiotics
are administered after such treatment.
Case 2
[0186] Dog R had a history of bilateral otitis, which had failed to
resolve despite treatment with gentamicin, marbofloxacin (used for
the treatment of Pseudomonas otitis), ampicillin (used for other
bacterial infections) and rirnadyl (an anti-inflammatory). Dog R
was examined on Feb. 4, 2016. At this time, both ears were infected
with both Pseudomonas aeruginosa and with coliform bacteria. Both
ears were producing purulent discharges accompanied by intense
odour. Erythema and ulceration were also marked. Following
examination, dog R was treated with BioVet-PA (100,000 infectious
units of each of the six bacteriophages in 0.2 ml of diluent).
Analysis following treatment showed that two of the six
bacteriophages were replicating. This was accompanied by a fall in
the number of Pseudomonas bacteria present in the ear and by
improvement in clinical symptoms. Following a four day monitoring
period after bacteriophage treatment, dog R was treated with
Amoxicillin Clavulanate tablets (for coliform bacteria present in
the ear) and Canaural ear drops (containing diethanolamine
fusidate, framycetin sulphate, nystatin and prednisolone).
Pseudomonas aeruginosa present in the ears was known to be
partially resistant to aminoglycoside antibiotics such framycetin.
The other components of the Canaural formulation are not used
against Pseudomonas (diethanolamine fusidate is an antibiotic used
against other bacterial infections including colifrom bacteria,
nystatin is an antifungal agent; prednisolone is an
anti-inflammatory) Antibiotic sensitivity of the coliform infection
was unknown, but a course of ampicillin completed on Jan. 30, 2004
had failed to resolve the infection.
Result
[0187] Examination of dog R on Feb. 1, 2004 showed that clinical
symptoms had resolved completely in both ears. Erythema and
ulceration were absent, discharges were normal and no odour was
detected. This demonstrated the efficacy of antibiotic treatment
undertaken after application of bacteriophages in resolving the
clinical symptoms of infection when antibiotics and other chemical
agents had failed to clear the infection.
* * * * *
References