U.S. patent application number 11/189819 was filed with the patent office on 2005-11-24 for compositions containing bacteriophages and method of using bacteriophages to treat infections.
This patent application is currently assigned to Nymox Corporation. Invention is credited to Averback, Paul, Ghanbari, Hossein A..
Application Number | 20050260171 11/189819 |
Document ID | / |
Family ID | 21772776 |
Filed Date | 2005-11-24 |
United States Patent
Application |
20050260171 |
Kind Code |
A1 |
Ghanbari, Hossein A. ; et
al. |
November 24, 2005 |
Compositions containing bacteriophages and method of using
bacteriophages to treat infections
Abstract
Purified, host-specific, non-toxic, wide host range and virulent
bacteriophage preparations that are effective in killing bacterial
organisms in vivo are disclosed. Also disclosed are compositions
containing these bacteriophages, methods of making the
bacteriophage preparations and methods of treating bacterial
infections using the compositions. Methods of treating bacterial
infections using the compositions containing the bacteriophages in
combination with conventional antibiotics also are disclosed.
Inventors: |
Ghanbari, Hossein A.;
(Beaconsfield, CA) ; Averback, Paul;
(Beaconsfield, CA) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Nymox Corporation
|
Family ID: |
21772776 |
Appl. No.: |
11/189819 |
Filed: |
July 27, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11189819 |
Jul 27, 2005 |
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09583738 |
May 31, 2000 |
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6942858 |
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09583738 |
May 31, 2000 |
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08842653 |
Apr 15, 1997 |
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6121036 |
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60015663 |
Apr 15, 1996 |
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Current U.S.
Class: |
424/93.6 ;
435/235.1 |
Current CPC
Class: |
Y02A 50/30 20180101;
Y02A 50/401 20180101; C12N 2795/00032 20130101; Y02A 50/473
20180101; A61P 31/00 20180101; C12N 7/00 20130101; Y02A 50/481
20180101; A61K 35/76 20130101; Y02A 50/475 20180101 |
Class at
Publication: |
424/093.6 ;
435/235.1 |
International
Class: |
A61K 038/43; C12N
007/00 |
Claims
1-22. (canceled)
23. A method of treating a mammal suffering from bacterial
infection comprising administering to the mammal an effective
amount of a composition comprising: (a) a purified, host-specific,
non-toxic, wide host-range, and virulent bacteriophage preparation,
wherein: (1) the bacteriophage preparation consists essentially of
two or more bacteriophage strains, wherein each bacteriophage
strain is specific for the bacterial infection treated and is
selected against one of the group consisting of staphylococci,
hemophilia, helicobacter, mycobacterium, mycoplasmi, streptococci,
neisserii, klebsiella, enterobacter, proteus, bacteriodes,
pseudomonas, borrelii, citrobacter, escherichia, salmonella,
propionibacterium, treponema, shigella, enterococci, and
leptospirex; (2) at least two of the bacteriophage strains are
isolated against different strains of bacterial organisms; and (3)
each bacteriophage strain is effective in killing, in vitro,
bacteria from at least about 50% of bacterial isolates, wherein the
isolates are from the same strain of bacterial organism as that
from which the bacteriophage strain is isolated; and (4) the
bacteriophage preparation can be safely administered to patients or
mammals in need; and (b) a pharmaceutically acceptable carrier.
24. The method of claim 23, wherein the bacterial organisms are
selected from the group consisting of Staphylococcus aureus,
Staphylococcus epidermidis, Helicobacter pylori, Streptococcus
pneumoniae, Streptococcus mutans, Streptococcus oralis,
Streptococcus parasanguis, Streptococcus pyogenes, Streptococcus
viridans, Group A streptococcus and anaerobic streptococcus,
Hemophilus influenzae, Shigella dysenteriae, Mycobacterium
tuberculosis, Mycobacterium leprae, Mycobacterium asiaticum,
Mycobacterium intracellulare, Mycoplasma pneumoniae, Mycoplasma
hominis, Neisseria meningitidis, Neisseria gonorrhea, Klebsiella
pneumoniae, Pseudomonas aeruginosa, Propionibacterium acnes,
Treponema pallidum, Treponema pertanue, Treponema carateum,
Escherichia coli, Salmonella typhimurium, Borrelia burgdorferi,
Leptospirex hemoragia, Klebsiella oxytoca, and Citrobacter
fruendii.
25. The method of claim 24, wherein at least one of the bacterial
organisms is Staphylococcus aureus.
26. The method of claim 24, wherein at least one of the bacterial
organisms is Streptococcus pyogenes.
27. The method of claim 24, wherein at least one of the bacterial
organisms is Citrobacter freundii.
28. The method of claim 24, wherein at least one of the bacterial
organisms is Klebsiella oxytoca.
29. The method of claim 24, wherein at least one of the bacterial
organisms is Escherichia coli.
30. The method of claim 24, wherein at least one of the bacterial
organisms is Salmonella typhimurium.
31. The method of claim 24, wherein the bacteriophage preparation
is resistant to one or more properties selected from the group
consisting of: (a) resistant to exposure to high temperatures; (b)
resistant to exposure to drying; (c) resistant to exposure to lytic
agents; (d) resistant to exposure to mutator hosts; (e) resistant
to heat shock; and (f) resistant to ionic variation.
32. The method of claim 23, further comprising administering an
antibiotic.
33. The method of claim 32, wherein the antibiotic is selected from
the group consisting of aminoglycosides, cephalosporins,
macrolides, erythromycin, monobactams, penicillins, quinolones,
sulphonamides, and tetracycline.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to the use of bacteriophages
to treat infectious diseases whereby the host-specific, wide
host-range bacteriophages are present, in purified form, in
non-toxic sufficiently virulent compositions that can be
administered to patients or mammals in need thereof. The
bacteriophages are useful in treating infections due to bacterial
microorganisms by killing a sufficient quantity of the bacterial
microorganisms or by rendering the bacterial microorganisms more
susceptible to other chemotherapeutic antibiotics.
[0003] 2. Description of Related Art
[0004] Bacteriophages (phages) are a heterogeneous group of viruses
that infect bacteria that were first discovered in the early part
of the 20th century. d'Herelle, F., The bacteriophage. Its role in
immunity, translated by Smith, G. H., Williams & Wilkins Co.,
Baltimore (1922). Bacteriophages presently are widely used in
scientific research, such as in molecular biology (e.g. as genetic
vectors) and in medical diagnostics (e.g. phage typing of
bacteria). Insofar as phages naturally infect and kill bacteria, it
traditionally was suggested that they could be utilized in medical
therapeutics, because bacteria are a major cause of disease.
[0005] Indeed, potential therapeutic uses of phage in experimental
systems have been reported extensively in the literature
(d'Herelle, F., The bacteriophage and its clinical applications,
Charles C. Thomas, Baltimore (1930)). None of these potential
experimental therapeutic uses has resulted in the formulation of an
efficacious bacteriophage preparation, i.e., one that is
sufficiently virulent, non-toxic, host-specific, and with wide
enough host range to be of practical use. The notion of phage
therapeutics has not resulted in practical usage because: (i)
efficacy has been marginal or non-existent; (ii) toxic side effects
of phage therapy have been unacceptable; (iii) better alternatives
such as conventional chemical antibiotics have existed; (iv)
conventional bacteriophage preparations are contaminated with
debris from bacterial lysis which typically contain toxins; (v) an
antibody response to the bacteria can be initiated upon
introduction of the phage; and (v) phage preparations do not arrive
at the target site because they are removed too fast from the body
once injected or ingested. Thus, with respect to phage therapeutics
in the use of ameliorating or treating specific animal infections,
there is no currently known practical workable concept or method.
Ostensibly, therefore, there is practically no known specifically
useful phage therapeutic or composition of matter containing a
phage therapeutic. In addition to the above general limitations,
there also have been little or no reported advantages-for the use
of phage therapeutics such as, for example, (i) useful innovations
for administration of phage therapy, (ii) adjunctive therapeutic
use of phage therapy with other antibiotherapeutics, or (iii) for
use in the context of microorganism resistance to conventional
therapies.
[0006] Despite the recognized value and importance of new
antimicrobial therapies, the potential of phage therapy has not
been accomplished in any practical sense that is used in modern
therapeutics, nor have methods, compositions, or other uses been
defined for nontoxic and efficacious therapies of this type with
practical and effective delivery. Phage preparations known in the
art and available today are not known to be effective in treating
mammalian bacterial infections (AMERICAN TYPE CULTURE COLLECTION
CATALOGUE OF BACTERIA AND BACTERIOPHAGES, (ATCC) 18th Edition,
pages 402-411 (1992). It is believed that conventional, or
wild-type phage preparations are: (i) incapable of surviving for
sufficient periods of time in vivo; (ii) impure in that they
contain various strains of bacteriophage including mutated phage
and the like which are not virulent to the respective bacterial
microorganism, as well as a host of bacterial impurities; (iii)
toxic; and (iv) not host-specific in that they do not tend to
migrate primarily to the bacterial infections.
[0007] Recent approaches have attempted to make use of phage
therapeutics by utilizing bacteriophages that are selected using a
specific serial passage method to produce longer circulating
bacteriophages. Specific bacteriophages have been developed to
Escherichia coli and Salmonella typhimurium by first isolating
bacteriophages and mutants thereof that are specific to these
bacterial microorganisms, and then purifying these bacteriophages
and selecting the strains that are capable of avoiding entrapment
in the reticuloendothelial system of the animal by consecutive
cycles of injecting the phage into the animal, isolating the phage
in the blood of the animal and regrowth of the phage in the
bacteria. The use of this serial passage method in ascertaining
efficacious bacteriophage, however, is extremely costly and time
consuming to develop and it is of unknown usefulness in the
majority of infections. Furthermore, a longer circulating
bacteriophage is not precluded from being less virulent and
therefore ineffective as a potential or theoretical treatment. This
method of developing bacteriophage preparations therefore is
undesirable, and the selection criteria utilized likely will not
yield a virulent, non-toxic host-specific bacteriophage
preparation.
[0008] One of the primary reasons why bacteriophages have not been
utilized to treat bacterial infections and bacterial microorganisms
is due to the widespread use of antibiotics. Numerous strains of
bacteria and microorganisms have evolved, however, which are
resistant to conventional antibiotics. There exists a need,
therefore, to develop methods of treating bacterial infections that
are resistant to conventional antibiotics or to develop a method
that can complement the antibiotherapy.
[0009] In addition, there exists a need to develop new and useful
phage therapeutics which can be used to treat infectious disorders
caused by bacterial microorganisms. There also exists a need to
develop compositions including phage therapeutics in
pharmaceutically acceptable vehicles that can be administered to
subjects infected with bacterial microorganisms. There is an
additional need to develop a method that is capable of delineating
useful phage therapeutics that is efficient and economically
feasible. There also exists a need to develop new and useful phage
therapeutics that are useful in combination with antibiotics to
treat infectious disorders caused by bacterial microorganism
infection. Finally, there exists a need to develop a bacteriophage
preparation that is host-specific, virulent and non-toxic thereby
rendering it useful in treating bacterial infections.
[0010] Examination of past failures with phage therapeutic attempts
and modern approaches to these failures, have now revealed that
phages used for antibacterial therapies in certain specific
situations are highly important which make this new type of
treatment very attractive and useful. The bacteriophage
preparations, however, must be selected so that virulent,
non-toxic, host-specific preparations are formulated. Thus, there
is a need to develop host-specific, non-toxic and virulent
bacteriophage preparations that can be used to effectively treat
bacterial infections in a mammal.
SUMMARY OF THE INVENTION
[0011] It is therefore an object of the present invention to
develop new and useful phage therapeutics which can be used to
treat infectious disorders caused by bacterial microorganism
infections. Another object of the invention is to provide a
bacteriophage preparation that is virulent, non-toxic and
host-specific. It is an additional object of the present invention
to provide a method of screening effective phage therapeutics that
is efficient and economically feasible. It is a further object of
the present invention to provide new and useful phage therapeutics
that can be used in combination with antibiotics to treat
infectious disorders. It is yet another object of the present
invention to provide a method of treatment using phage therapeutics
in novel vehicle preparations for treating animal infections caused
by microorganisms.
[0012] In accordance with these objectives, there is provided a
composition comprising a microorganism host specific non-toxic
purified phage preparation specific to a microorganism including,
inter alia, staphylococci, hemophilii, helicobacter, mycobacterium,
mycoplasmi, streptococci, neisserii, klebsiella, enterobacter,
proteus, bacteriodes, pseudomonas, borrelii, citrobacter,
escherichia, salmonella, propionibacterium, treponema, shigella,
enterococci and leptospirex. Preferably the micoorganism includes,
inter alia, Staphylococcus aureus, Staphylococcus epidermidis,
Helicobacter pylori, Streptococcus pneumoniae, Streptococcus
mutans, Streptococcus oralis, Streptococcus parasanguis,
Streptococcus pyogenes, Streptococcus viridans, Group A
streptococcus and anaerobic streptococcus, Hemophilus influenzae,
Shigella dysenteriae, Mycobacterium tuberculosis, Mycobacterium
leprae, Mycobacterium asiaticum, Mycobacterium intracellulare,
Mycoplasma pneumoniae, Mycoplasma hominis, Neisseria meningitidis,
Neisseria gonorrhea, Klebsiella pneumoniae, Pseudomonas aeruginosa,
Propionibacterium acnes, Treponema pallidum, Treponema pertanue,
Treponema carateum, Escherichia coli, Salmonella typhimurium,
Borrelia burgdorferi, Leptospirex, such as Leptospirex hemoragia
Citrobacter fruendii, and the like. There also are provided
compositions comprising the aforementioned phage preparations
either in aqueous injectable form or in the form of liposomes, a
topical preparation, a transdermal patch or other capsule which may
obviate administration by injection.
[0013] In accordance with an additional object of the present
invention, there is provided a method of screening efficacious
phage therapeutics by selecting phages that are more virulent and
more adaptable in vivo thereby rendering them more capable of
survival in vivo and more capable of infecting and killing
bacteria. In accordance with this object of the present invention,
bacteriophages can be selected which: (i) have a higher frequency
per unit volume of foci of bacterial killing (plaque forming units
per volume); (ii) have a higher frequency per unit volume of foci
of bacterial killing with a smaller volume of phage preparations;
and (iii) are capable of killing a wider range of host bacteria
from a wider range of different isolated cultures of a given
bacteria, i.e., a wide host range. Other techniques useful for
selecting bacteriophage include selecting those that: (i) are
resistant to exposure to high temperatures or drying; (ii) are
resistant to exposure to lytic agents or mutator hosts; (iii)
survive for a period of time greater than 24 hours under normal or
abnormal conditions; (iv) are resistant to heat shock; and/or (v)
are resistant to ionic variation including drying, overhydration or
extremes of individual ionic concentrations.
[0014] In accordance with an additional object of the invention,
there is provided a method of making a host-specific, non-toxic and
virulent bacteriophage preparation whereby the preparation includes
a bacteriophage that is effective in killing bacterial organisms in
vivo. The method includes obtaining a sample containing a
bacteriophage to at least one bacterial organism selected from
staphylococci, hemophilii, helicobacter, mycobacterium, mycoplasmi,
streptococci, neisserii, klebsiella, enterobacter, proteus,
bacteriodes, pseudomonas, borrelii, citrobacter, escherichia,
salmonella, propionibacterium, treponema, shigella, enterococci and
leptospirex. The sample then is dispersed in phosphate buffered
saline, filtered through a filter that will retain the bacterial
organism and allow the bacteriophage to pass and the resulting
bacteriophage is purified. Bacteriophage then are grown in a medium
containing at least one of the bacterial organisms, and
bacteriophage preparations achieving titers higher than about
10.sup.8 to 10.sup.9 bacteriophages per plaque after about eight
hours are selected and isolated to provide an isolated
bacteriophage. The isolated bacteriophage then is purified and the
purified bacteriophage is again grown in a medium containing at
least one of the bacterial organisms. This procedure of growing the
bacteriophage in a medium containing at least one of the bacterial
organisms, selecting, isolating and purifying the bacteriophage
then is repeated anywhere from about 5 to about 25 times, and a
bacteriophage that is virulent and capable of killing a wide host
range of bacterial organisms is selected, isolated and purified to
prepare a host-specific, non-toxic and virulent bacteriophage
preparation.
[0015] In accordance with an additional object of the present
invention, there is provided a method of making a host-specific,
non-toxic virulent bacteriophage preparation whereby the
preparation includes a bacteriophage that is effective in killing
bacterial organisms in vivo. The method includes obtaining a sample
containing a bacteriophage to at least one bacterial organism
listed above, dispersing the sample in saline, filtering through a
filter that will retain the bacterial organism and allow the
bacteriophage to pass and the resulting bacteriophage is purified.
The purified bacteriophage then are grown in a medium containing at
least one of the bacterial organisms, and bacteriophage
preparations achieving titers higher than about 10.sup.8 to
10.sup.9 bacteriophages per plaque after about eight hours are
selected and isolated to provide an isolated bacteriophage. This
procedure can be repeated once, twice or up to 30 times, if desired
to produce a purified and isolated bacteriophage. The purified and
isolated bacteriophage then is subjected to at least one mutating
condition selected from mutator host, irradiation, temperature and
pH extremes, ionic variation, drying or overhydration, extreme
ionic concentration and heat shock, whereby at least one
bacteriophage survives the mutating condition. The bacteriophage
that survives then is isolated and purified to prepare a
host-specific, non-toxic and virulent bacteriophage
preparation.
[0016] In accordance with another object of the present invention,
there is provided a method of treating a mammal infected with a
microorganism selected from any of the microorganisms listed above
whereby the method includes administering to the mammal a
therapeutically effective amount of the aforementioned composition.
In accordance with yet another object of the present invention,
there is provided a method of treating a mammal infected with the
aforementioned microorganisms comprising administering to the
mammal a therapeutically effective amount of the aforementioned
composition in combination with an antibiotic. In accordance with
these methods, the aforementioned compositions can be administered
by routes including but not limited to intravascular injection,
oral, intranasal, topical, sub- trans- and intracutaneously, and by
other routes.
[0017] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 represents an electron micrograph
(magnified.times.450,000), of 173A E. coli bacteriophage,
negatively stained, from Example 6.
[0019] FIG. 2 represents an electron micrograph
(magnified.times.225,000) of 146A E. coli bacteriophage, negatively
stained, from Example 6.
[0020] FIG. 3 represents DNA fingerprinting of 173A E. coli
bacteriophage of Example 6.
[0021] FIG. 4 represents DNA fingerprinting of 146A E. coli
bacteriophage of Example 6.
[0022] FIG. 5 represents an electron-micrograph
(magnified.times.225,000) of 262A C. fruendii bacteriophage,
negatively stained, from Example 7.
[0023] FIG. 6 represents DNA fingerprinting of 174A K. oxytoca
bacteriophage of Example 7.
[0024] FIG. 7 represents DNA fingerprinting of 83A S. aureus
bacteriophage of Example 9.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0025] Throughout this description, the expression "wide host
range" denotes a bacteriophage that is capable of killing bacteria
from a variety of different hosts. Preferably, the bacteriophage of
the present invention are capable of killing bacteria from at least
about 50% of the host samples. Throughout this description, the
term "virulent" denotes a bacteriophage that is capable of
effectively killing bacteria from a wide host range. Preferably,
the bacteriophage that are selected are effective in killing about
100% more bacteria from various sources, or hosts, when compared to
the bacteriophage that are not selected. More preferably, the
bacteriophage are selected that kill about 200% more bacteria, and
most preferably, bacteriophage are selected that kill about 300%
more bacteria. For example, by means of illustration only, assume
that two bacteriophage preparations are selected and each are
tested against bacteria samples from 100 different hosts. If the
first one is effective in killing bacteria from 15 hosts, and the
second is effective in killing bacteria from 60 hosts, then the
second bacteriophage preparation would be virulent and would have a
wider host range, and the second bacteriophage preparation would
killing 300% more bacteria than the first.
[0026] "Virulent bacteriophage preparations" also denotes
bacteriophage preparations that are capable of killing bacteria at
a lower concentration than non-selected non-virulent bacteriophage.
For example, and for purposes of illustration only, assume that two
bacteriophage preparations are selected and the first one is
effective in killing bacteria at 10.sup.6 dilution. If the second
phage preparation is effective in killing bacteria at 10.sup.8
dilution, then the second phage preparation can be said to be
virulent, when compared to the first. The expression "non-selected
bacteriophage" denotes bacteriophage preparations that are prepared
together with the virulent, host-specific, purified, wide host
range and non-toxic bacteriophage of the present invention, but are
not selected because they are less virulent, and/or are less
host-specific, and/or have a much narrower host-range, and/or are
toxic and/or are not purified. Hence, the bacteriophage
preparations prepared and selected in accordance with the present
invention are even more superior, when compared to the non-selected
bacteriophage, in terms of virulence, host-specificity, host range,
toxicity and purity than commercially available, or wild-type
bacteriophage preparations that are available from the Center for
Disease Control (CDC) or the ATCC.
[0027] In accordance with the present invention, it has been
determined that host-specific, virulent and non-toxic bacteriophage
preparations can be used to treat specific bacterial infections.
Particularly effective in this regard are host-specific, non-toxic
and purified phage preparations which act by killing the
microorganism, and which act on specific infections by obliterating
sufficient numbers of the microorganisms present in the infectious
focus. Throughout this description, the expression "purified"
denotes a phage preparation that contains substantially no toxins,
(no endotoxins) preferably less than 1.0% by weight of toxins
(endotoxins) and more preferably less than about 0.05% by weight of
toxins (endotoxins). Traditional bacteriophage preparations that
were known to be inefficacious in vivo were not host-specific,
typically were not purified, usually were toxic and typically were
not virulent in vivo. The bacteriophage preparations according to
the present invention, however, have alleviated these problems in
that the inventive phage preparations are selected, using the
guidelines provided herein, to be highly host specific, non-toxic
and purified to the extent that renders the inventive phage
preparations effective in killing and/or obliterating and/or
reducing sufficient numbers of host bacteria in vivo.
[0028] Host-specific, non-toxic, wide host range, virulent and
purified phage preparations also can be used that reduce but do not
entirely obliterate the population of microorganisms, thereby
rendering the infectious focus more susceptible to other
chemotherapeutic antibiotics and thus reducing in combination
therapy duration, side effects, and risks of the latter. Thus, the
phage preparations in this embodiment of the invention preferably
can be used in combination with known antibiotics such as
aminoglycosides, cephalosporins, macrolides, erythromycin,
monobactams, penicillins, quinolones, sulfonamides, tetracycline,
and various anti-infective agents. Those skilled in the art can
refer to the Physician's Desk Reference, 50th Ed (Medical Economics
(1996)), or similar reference manuals for a more complete listing
of known antibiotics which could be used in combination with the
inventive phage preparations. For example, a phage preparation
effective against various strains of staphylococcus could be used
in combination with a cephalosporin such as Keflex.RTM. or
Keftab.RTM. (both from Cephalexin). Those skilled in the art, using
the guidelines provided herein, are capable of designing an
effective treatment regimen by either using the phage preparation
alone or using a phage preparation in combination with
antibiotics.
[0029] The host-specific, non-toxic and purified phage preparations
of the invention can be prepared by growing in the presence of a
microorganism including, inter alia, staphylococci, hemophilii,
helicobacter, mycobacterium, mycoplasmi, streptococci, neisserii,
klebsiella, enterobacter, proteus, bacteriodes, pseudomonas,
borrelii, citrobacter, escherichia, salmonella, propionibacterium,
treponema, shigella, enterococci and leptospirex. Preferably the
micoorganism includes, inter alia, Staphylococcus aureus,
Staphylococcus epidermidis, Helicobacter pylori, Streptococcus
pneumoniae, Streptococcus mutans, Streptococcus oralis,
Streptococcus parasanguis, Streptococcus pyogenes, Streptococcus
viridans, Group A streptococcus and anaerobic streptococcus,
Hemophilus influenzae, Shigella dysenteriae, Mycobacterium
tuberculosis, Mycobacterium leprae, Mycobacterium asiaticum,
Mycobacterium intracellulare, Mycoplasma pneumoniae, Mycoplasma
hominis, Neisseria meningitidis, Neisseria gonorrhea, Klebsiella
pneumoniae, Pseudomonas aeruginosa, Propionibacterium acnes,
Treponema pallidum, Treponema pertanue, Treponema carateum,
Escherichia coli, Salmonella typhimurium, Borrelia burgdorferi,
Leptospirex, such as Leptospirex hemoragia Citrobacter fruendii.
More preferably, the microorganism is selected from any one of
staphylococci, streptococci, citrobacter, escherichia and
klebsiella, and most preferably, the microorganism is selected from
Staphylococcus aureus, Streptococcus pyogenes, Klebsiella oxytoca,
Escherichia coli and Citrobacter fruendii.
[0030] The phages employed according to the present invention can
be any phages capable of infecting and killing bacteria and
bacterial infections in mammals. Preferably, the phages of the
invention can initially be derived from microorganisms by standard
methods well known to those skilled in the art or can be obtained
from various depositories. AMERICAN TYPE CULTURE COLLECTION
CATALOGUE OF BACTERIA AND BACTERIOPHAGES, 18th Edition, pages
402-411 (1992). Selection of phage for virulence and persistence
then can be effected by methods such as repeated growth in bacteria
followed by selection, isolation and purification of phage from
plaque forming units, as well as selection based on resistance to
physical methods such as temperature and/or drying, or by
biochemical-toxic methods such as exposure to lytic agents or
mutator hosts, or by longevity of survival under normal or abnormal
conditions. After culture with or without selection for virulence,
specific phage can be purified to homogeneity. This can be
accomplished by filtration, plaque formation, and analytical
density gradient ultracentrifugation in gradients such as sucrose,
Ficoll, Percoll or cesium chloride. Those skilled in the art are
capable of selecting and purifying a given phage preparation using
the guidelines provided herein, coupled with known purification
techniques.
[0031] To be effective in killing and/or obliterating bacterial
microorganisms, or to be capable of reducing sufficient quantities
of these microorganisms, the bacteriophages of the present
invention are subjected to the above-mentioned selection methods to
specifically select the virulent bacteriophage. Phage preparations
known in the art and available today are not known to be effective
in treating mammalian bacterial infections (ATCC CATALOGUE OF
BACTERIA AND BACTERIOPHAGES, 18th Edition, pages 402-411 (1992).
While not intending to be bound by any theories, it is believed
that conventional phage preparations are: (i) incapable of
surviving for sufficient periods of time in vivo; (ii) impure in
that they contain various strains of bacteriophage including
mutated phage and the like which are not virulent to the respective
bacterial microorganism, as well as a host of bacterial impurities;
(iii) toxic; and (iv) not host-specific in that they do not tend to
migrate primarily to the bacterial infections. The phage
preparations of the present invention do not suffer from these
drawbacks, however, because the inventive phage preparations are
selected for virulence and specificity, and are purified and
concentrated. The inventive phage preparations therefore are
efficacious in vivo in killing, obliterating or reducing sufficient
quantities of bacterial microorganisms.
[0032] Various mechanisms can be used to select bacteriophage
preparations for virulence. Initially, however, bacteriophage
preparations must be derived from microorganisms, which can be
effected using methods known in the art. (See, AMERICAN TYPE
CULTURE COLLECTION CATALOGUE OF BACTERIA AND BACTERIOPHAGES, 18th
Edition, pages 402-411 (1992). For example, samples can be
collected from individuals who suffer from a bacterial infection.
Various samples can be taken from various places on the body
including the throat, blood, urine, feces, spinal fluid, nasal
mucosa, skin, washings from the larynx and trachea, and the like.
Sample sites can be selected depending upon the target organism.
For example, a throat swab likely would be used to collect a sample
of a given strain of streptococcus, a skin culture likely would be
used to collect a sample of a given strain of staphylococcus, a
spinal fluid or blood sample likely would be used to collect a
sample of Neissereia meningitidis, a urine sample can be used to
collect samples of E. coli, and the like. Those skilled in the art
are capable of obtaining an appropriate sample from the respective
locus, given the target organism. Alternatively, bacterial strains
can be obtained from various laboratories including those available
from the National Institutes for Health (NIH), the ATCC and the
like.
[0033] Preferably, samples are obtained from various medical
professionals, the samples are purified, and colonies of the
bacteria are grown using conventional methods. When a bacterial
organism is identified from any of the aforementioned sources, it
preferably is grown in pure culture and frozen for storage at about
-20.degree. C., -70.degree. C. and -80.degree. C. These organisms
can be frozen using conventional methods. A particularly preferred
method for freezing bacterial samples includes preparing overnight
cultures of bacterial isolates, and then adding about 400 .mu.l of
a sterile 80% glycerol to about 1 ml of overnight culture. The
microtubes then can be labeled and stored at -20.degree. C.,
-70.degree. C. and -80.degree. C.
[0034] These samples then can be used to generate the bacteriophage
preparations by dispersing the sample in a solution, for example, a
phosphate buffered saline solution, and then filtering the solution
through a very small pore size filter to retain the target organism
and permit the bacteriophage to pass through. Preferably, a filter
having a pore size in the range of from about 0.01 to 1 .mu.m can
be used, more preferably, from about 0.1 to about 0.5 .mu.m and
most preferably from about 0.2 to about 0.4 .mu.m. Bacteriophage
preparations then can be generated by growing in a medium
containing the target organism with periodic titering. The
bacteriophage samples are taken periodically, preferably about
every 2 hours and the quantity of bacteriophage titered. Those
skilled in the art are capable of growing bacteriophage in the
bacterial host using conventional methods such as those described
in, inter alia, Silhavy et al., EXPERIMENTS WITH GENE FUSION, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984).
[0035] When a sufficient quantity of bacteriophage is achieved,
preferably about 10.sup.5 to about 10.sup.10, usually about
10.sup.8 to about 10.sup.9, the phage preparations can be isolated
using filtration with filters such as those described above, and
the samples can be purified by centrifugation, for example, using
cesium chloride density centrifugation as described in Sambrook et
al., MOLECULAR CLONING: A LABORATORY MANUAL, 2.sup.nd Edition, Cold
Spring Harbor, N.Y., pp. 2.73-2.81 (1989). Usually, the
bacteriophage preparations achieving higher titers at earlier
sampling times are selected and separated. Those skilled in the art
are capable of selecting and separating the appropriate
bacteriophage using the guidelines presented herein. Preferably,
this method of growing the phage in a medium containing the
bacteria, and then selecting, isolating and purifying phage from
plaque forming units is repeated a number of times until a virulent
phage preparations is obtained. Most preferably, the method is
repeated from about 5 to about 25 times.
[0036] Upon separating, or while growing in the medium together
with the host or target organism, the bacteriophage also can be
selected for virulence by subjecting it to various stringent
conditions. For example, the phage preparation can be subjected to
mutating conditions using a mutator host. Alternatively, the phage
preparation medium can be subjected to irradiation by light and/or
heat, it can be subjected to extreme variations in pH by addition
of appropriate acid or caustic, the medium can be subjected to
overhydration or to drying conditions, extreme individual ionic
concentration or heat shock. These various procedures are conducted
on the phage medium to assure bacteriophage diversity and to
increase the probability of selecting more virulent and more in
vivo adaptable bacteriophages. Those bacteriophages that survive
the aforementioned conditions then are separated using the methods
described above, and purified. If desired, additional bacteriophage
can be generated or cloned using techniques known to those skilled
in the art, and the resulting preparations purified. Skilled
artisans are capable of carrying out the aforementioned testing
methodology to select virulent phage preparations, using the
guidelines provided herein, and also are capable of separating and
purifying the virulent phage preparations that survive any and/or
all of the aforementioned testing protocol.
[0037] These bacteriophage preparations then optionally can be
administered to a test animal, for example, a mouse, by injection
and samples can be withdrawn at various sites (i.e., skin samples,
throat swabs, nasal mucosa samples, rectal samples, blood samples
and the like). The bacteriophage samples that are the most virulent
can be determined by plating by using standard methods. The
bacteriophage samples that are the most virulent then can be
isolated, grown, purified and detoxified, if needed.
[0038] These purified bacteriophage preparations then can be
administered to a test animal that has been infected with the
respective bacterial microorganism. The virulence of the
bacteriophage preparations can be determined by comparing the
microbial counts of bacteriophage from infected animals versus the
microbial counts for non-infected, or control, animals. The
bacteriophage preparations having the highest anti-bacterial
activity then can be selected, isolated and purified. The resulting
bacteriophage preparation then can be detoxified using techniques
known in the art.
[0039] For example, the purified bacteriophage preparation can be
subjected to ultrafiltration through a membrane or hollow fiber
with a molecular weight cut-off of approximately 10.sup.4 to about
10.sup.7 daltons, preferably within the range of from about
10.sup.5 to about 10.sup.6 daltons. Alternatively, the phage
preparations can be subjected to ultracentrifugation using
preparations such as cesium chloride (i.e., at a concentration
range of from about 60 to about 95 grams per mole), percoll,
ficoll, (i.e., at about 50 to about 80%) or sucrose and glycerol
(i.e., at about 5% to about 40%). Ultracentrifugation can be
carried out at forces within the range of from about 50,000.times.g
to about 90,000.times.g, preferably from about 60,000.times.g to
about 80,000.times.g, for 2 or more hours for cesium chloride,
sucrose and glycerol, or for example, from about 20,000.times.g to
about 40,000.times.g, preferably from about 25,000.times.g to about
35,000.times.g for 20 to 30 minutes using percoll or ficoll.
[0040] In addition, the phage preparations can be detoxified by
dialysis using the largest pore membrane that retains
bacteriophages, where the membrane preferably has a molecular
weight cut-off of approximately 10.sup.4 to about 10.sup.7 daltons,
preferably within the range of from about 10.sup.5 to about
10.sup.6 daltons. Alternatively, the phage preparations can be
detoxified using size exclusion chromatography using preparatory
columns with an exclusion pore of any where from about 10.sup.4 to
about 10.sup.7 daltons, preferably from about 10.sup.5 to about
10.sup.6 daltons.
[0041] These purified phage preparations which have been selected
for virulence then can be formulated into compositions that can be
administered in large enough dosage and in adequate frequency to
effect arrest or retardation of infection. The above durations and
dosages can be determined on a specific basis for each specific
type of phage in each specific type of infection in each specific
host. Thus, those skilled in the art will recognize that the
effective dosage in a mouse will differ from that of a man, and
will also vary according to the specific microorganism. The
quantitative determinations can be readily determined by one of
ordinary skill in the art.
[0042] The compositions of the present invention can be prepared by
admixing a quantity of purified phage preparation with a
pharmaceutically acceptable carrier. Usually, the compositions of
the present invention are advantageously administered in the form
of injectable compositions. A typical composition for such purpose
comprises a pharmaceutically acceptable carrier. For instance, the
composition may contain about 10 mg of human serum albumin and from
about 20 to 200 micrograms of the phage preparation per milliliter
of phosphate buffer containing NaCl. Other pharmaceutically
acceptable carriers include aqueous solutions, non-toxic
excipients, including salts, preservatives, buffers and the like,
as described in REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed.
Easton: Mack Publishing Co. pp 1405-1412 and 1461-1487 (1975) and
THE NATIONAL FORMULARY XIV., 14th Ed. Washington: American
Pharmaceutical Association (1975), the contents of which are hereby
incorporated by reference. Examples of non-aqueous solvents include
propylene glycol, polyethylene glycol, vegetable oil and injectable
organic esters such as ethyloleate. Aqueous carriers can include
water, alcoholic/aqueous solutions, saline solutions, parenteral
vehicles such as sodium chloride, Ringer's dextrose, etc.
Intravenous vehicles include fluid and nutrient replenishers.
Preservatives include antimicrobials, anti-oxidants, chelating
agents and inert gases. The pH and exact concentration of the
various components of the bacteriophage compositions of the
invention can be adjusted according to routine skill in the art.
See GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS
(7th ed.).
[0043] Advantageously, the bacteriophage compositions of the
present invention can be in the form of liposomes, lipophilic
microcapsules, dendrimers or the like for oral administration to
treat systemic infections. Those skilled in the art are capable of
preparing the phage preparations of the present invention in the
form of a lipophilic microcapsule, a dendrimer or a liposome using
conventional techniques known in the art. The skilled artisan also
is capable of providing a bacteriophage preparation composition
that can be administered intranasally, rectally, transdermally,
topically, or other known routes of administration of
medicaments.
[0044] The compositions of the present invention can be used to
treat mammals having bacterial infections. Specifically, the
compositions of the present invention are useful in treating
mammals infected with any of the bacterial microorganisms listed
above. Suitable bacteriophage-containing compositions can be
prepared that will be effective in killing, obliterating or
reducing the quantity of any of the aforementioned bacterial
microorganisms using the guidelines presented above.
[0045] The compositions of the present invention preferably are
administered intravenously, intranasally, orally, etc., in an
amount and for a period of time effective to treat the bacterial
infection. The expression "treating bacterial infections," as it is
used throughout this description, denotes either (i) killing or
obliterating sufficient bacterial microorganisms to render the
microorganisms ineffective in infecting the host, or (ii) reducing
a sufficient quantity of bacterial microorganisms so as the render
the microorganisms more susceptible to treatment using conventional
antibiotics. Determining an effective amount of host-specific,
non-toxic purified phage preparation to be administered in
accordance with the present invention entails standard evaluations.
An assessment in this regard would generate data concerning
bioavailability, absorption, metabolism, serum and tissue levels
and excretion, as well as microorganism levels, markers, and
cultures. The appropriate dosage and duration of treatment can be
ascertained by those skilled in the art using known techniques.
[0046] The present invention now will be illustrated by the
following examples which are intended to further exemplify
particularly preferred embodiments of the invention, and are not
meant to limit the invention.
[0047] The wild-type bacteriophage samples obtained from publicly
available sources such as the ATCC or CDC are not suitable for
therapeutic use because they are not non-toxic, host-specific,
virulent, purified or they do not have a wide host range. In
contrast, bacteriophage preparations prepared in accordance with
the foregoing description and following examples are suitable for
therapeutic use because they are non-toxic, host-specific,
virulent, purified and have a wide host range.
EXAMPLES
Example One
[0048] The following procedure can be followed to prepare purified,
toxin-free phage preparations specific to any of the bacterial
microorganisms listed above. Bacteriophage specific to any of these
microorganisms can be grown in vitro or in vivo in a test animal
such as a mouse using methods known in the art. Wood, W. B., et
al., Building a Bacterial Virus, Scientific American, (1967).
Samples of materials containing the bacteriophage then are
collected and further treated to isolate and purify the
bacteriophage.
[0049] The phage purification and toxin removal will be
accomplished by differential centrifugation, serial filtration and
size exclusion chromatography. Toxins are removed thoroughly by
surfactants of high non-ionic strength as well as dissociating
compounds (urea and sucrose). Commercial kits are used to monitor
toxin contamination. (Limulus Amebocyte Lysate Assay, Associates of
Cape Cod Inc., Woods Hole Mass.).
Example Two
[0050] The purified phage preparation suitable for oral
administration to treat systemic infections is prepared by
packaging the phage preparation into highly lipophilic
microcapsules such as liposomes or dendrimers. Delivery also is
effected by the transnasal route.
Example Three
[0051] The following procedure can be followed to determine the
clinical efficacy of the host-specific, non-toxic purified phage
preparation of Example 1, or the phage compositions of Example
2.
[0052] Test One
[0053] Test animals such as dogs, rats, monkeys or other species
are subjected to lethal doses of bacteria such as klebsiella or
enterobacter and are observed clinically and microbiologically.
Equal numbers of such animals subjected to the above lethal doses
also are treated with adequate dosages of the phage preparations of
Example 1 delivered by intravenous, intraperitoneal, intramuscular,
transcutaneous or other route by adequate vehicle such as liposome
or patch or other capsule obviating injection, and are observed
clinically and microbiologically.
[0054] Results: Results are assayed in clinical and microbiological
terms. Cultures from treated versus non-treated animals with
quantitative titers show that the host-specific non-toxic purified
phage preparations of Example 1 inhibits infection significantly.
Clinical examinations of treated versus untreated animals show that
the host-specific non-toxic purified phage preparations of Example
1 inhibits infection significantly. In untreated animals infected
with lethal dosages as above, progressive deterioration leading to
death is found in all or practically all cases. Progressive
deterioration often includes but is not limited to: fever,
lethargy, and fluid imbalance and secondary symptoms, and may
include hyperactivity, seizures, hemorrhage, and other
manifestations. In sharp contrast to the un-treated animals,
treated animals eliminate the bacteria and return to pre-infection
clinical status. Clinical evolution in treated cases depends on the
duration of pretreatment inoculation, which in turn determines
severity of pretreatment infection development.
[0055] Other assays of treatment efficacy are within the scope of
the present invention, such as non-microbiologic serologic and
hematologic tests such as erythrocyte sedimentation rate, blood
cell counts, antibody response, or radiological, scanning or other
procedures well known to health care and research personnel.
Example Four
[0056] The following method can be used for preparation of virulent
phage preparations for specific use in treating specific bacterial
infections. Experimental animals are infected with a specific
microorganism at a standard dosage and then cultures with higher
titer of phage are selected for phage usage. Alternatively,
cultures are tested for phage quantities and higher quantity phages
are selected, isolated and cultured. The isolated phage
preparations then are formulated into compositions for intravenous,
topical or other route of administration.
Example 5
[0057] The following example illustrates the isolation and
purification of phage preparations, and then the selection of
virulent phages that will be useful in effectively treating
bacterial infections in mammals.
[0058] Samples are collected from individuals from sample sites
such as throat swabs, blood samples, urinary samples, spinal fluid
samples, nasal swabs, rectal swabs, stool samples, skin scrapings,
sputum, washings from larynx and trachea, and other sources,
biopsies and other sites of infectious foci. These sample sites are
selected on the basis of the target bacterial microorganism such as
any of the bacterial microorganisms listed above. For example, a
throat swab is used to collect a sample of a strain of
streptococci, a spinal fluid or blood sample is used for collecting
a sample of Neisseria meningitidis, and a skin or abscess scraping
or biopsy or swab is used for collecting a sample of Staphylococcus
aureus.
[0059] The sample collected then is dispersed in phosphate buffered
saline, pH of about 7.0, and then filtered through a membrane
having a pore size ranging from 0.2 to 0.4 .mu.m, which will retain
the target bacterial organism and allow the bacteriophage to pass
through. The filtrate therefore will contain bacteriophages to the
target host microorganism.
[0060] Culture plates infected with the target bacterial organisms
are used to screen for bacteriophages by plaque formation by
standard methods.
[0061] Individual plaques then are purified and the bacteriophage
viruses grown in medium containing the targeted microorganism. The
mechanisms involved in the growth of bacteriophage in the presence
of a host bacteria is known in the art and described in, for
example, Stryer, Lubert, BIOCHEMISTRY, 3.sup.RD ED., W. H. Freeman
and Company, N.Y. (1988); Freifelder, David, MOLECULAR BIOLOGY,
2.sup.ND ED., Jones and Bartlett, Inc., MA (1987). The growth rate
is determined and documented by, for example, taking samples every
two hours and titering the quantity of bacteriophage. Bacteriophage
preparations achieving higher titers at earlier sampling times are
selected and isolated, typically, those achieving titers higher
than about 10.sup.8 to 10.sup.9 bacteriophages per plaque after
about eight hours.
[0062] Upon isolation, or during growth, mutating conditions
selected from mutator host, irradiation, temperature and pH
extremes, ionic variation of the medium including drying or
overhydration or extremes of individual ionic concentrations, and
heat shock are introduced in order to maximize bacteriophage
diversity (mutated bacteriophages add to the pool of bacteriophage
which are to be selected) and to enhance the probability of
selection of more virulent and more in vivo adaptable
bacteriophages.
[0063] The selected and isolated bacteriophage samples then are
subjected to an additional filtration through a filter having a
pore size within the range of from about 0.2 to about 0.4 .mu.m to
isolate the bacteriophages from the culture system. To assure
homogeneity of these isolated bacteriophage preparations, the steps
of (i) screening the bacteriophage for plaque formation, (ii)
growing bacteriophage in medium with the target microorganism, and
then (iii) subjecting the bacteriophage to mutating conditions are
repeated using the isolated bacteriophage preparations. The final
bacteriophage preparations are titered to determine the number of
plaque forming units in each preparation using methods known it the
art. Freifelder, D. supra.
[0064] These purified phage preparations then are further treated
and processed to remove endotoxins and other toxins using
procedures known in the art such as ultrafiltration,
ultracentrifugation, size exclusion chromatography and affinity
chromatography. A final preparation of bacteriophage then is
prepared by collating and mixing such that aliquots of the final
mixture contain equal numbers of the bacteriophages.
[0065] These final preparations then are administered by injection,
topical application or other means in mice, and samples from
various sites such as blood and spinal fluid samples, throat swabs,
nasal swabs, and the like are collected at various time intervals,
for example at six hours, 24 hours, 2 days, 3 days, 4 days and 5
days. The more virulent bacteriophages are identified by plating,
and these then are isolated, grown, purified and detoxified as
described above.
[0066] These bacteriophage preparations then are administered to
mice previously infected with the targeted bacterial microorganism,
and the virulence of the bacteriophage is determined by comparing
the microbial counts of treated versus non-treated animals over
time. The bacteriophages with the highest anti-bacterial activity
then is selected, isolated and purified. Toxin removal then can be
carried out using any of the following techniques: (i)
ultrafiltration through a membrane or hollow fiber with a molecular
weight cut-off of 10.sup.5 to 10.sup.6 daltons; (ii)
ultracentrifugation using preparations such as cesium chloride (60
to 95 g/100 ml), percoll or ficoll (50 to 80%), and sucrose or
glycerol (5 to 40%) at forces of 60,000.times.g to 80,000.times.g
for 2 hours (cesium chloride, sucrose and/or glycerol), or at
forces of 25,000.times.g to 35,000.times.g for 20 to 30 minutes
(percoll or ficoll); (iii) dialysis using the largest pore membrane
that retains bacteriophages with molecular weight cut-off of
10.sup.5 to 10.sup.6 daltons; and (iv) size exclusion
chromatography using preparatory columns with an exclusion pore of
10.sup.5 to 10.sup.6 daltons.
[0067] The purified and detoxified bacteriophages then are
formulated into compositions suitable for administration to a
mammal. A mammal suffering from a bacterial infection then can be
treated by administering the bacteriophage containing composition
in an amount and for a period of time effective to treat the
bacterial infection by killing the bacterial microorganisms which
caused the infection.
Example 6
[0068] Purified E. coli bacteriophage isolates were obtained from
sewage using the following procedure. About 100 .mu.l of sewage
filtrate (0.45 .mu.m acetate filters) was mixed with about 200
.mu.l culture of an E. coli isolate grown to saturation in LB
medium. Tubes were incubated at room temperature for about fifteen
minutes, and then 3.5 ml LB top agar (0.7% agar tempered to
50.degree. C.) was added to the tubes, vortexed, and poured onto
fresh LB plates. Plates then were incubated inverted at 37.degree.
C. overnight. Single plaques were chosen using a sterile Pasteur
pipette, and the agar plug was placed in a tube containing 1 ml
sterile SM buffer. One drop of chloroform was added, and the tube
was vortexed briefly. The solution then was allowed to stand for at
least one hour at room temperature for phage to elute from the
plaque. Phage then were re-plated at a low density (.about.10
plaques per plate). A single, well isolated plaque was chosen and
plated once more to produce a pure phage preparation.
[0069] To prepare high titer stock of phage preparations, about 100
.mu.l of phage stock prepared above (.about.10.sup.6 PFU) was added
to about 200 .mu.l of an E. coli isolate grown to saturation. Top
agar (3.5 ml LB; 0.3%) was added as described above, and poured.
Plates were incubated at 37.degree. C. overnight. If confluent
lysis was achieved, 3 ml of SM buffer was added, and buffer plus
overlay was scraped off using a bent glass rod and placed into a
sterile 50 ml Falcon tube. Several drops of chloroform were added
and the tubes were shaken gently for about ten minutes. Samples
then were spun at about 2800 rpm for twenty minutes, the
supernatant was removed and filtered (0.45 .mu.m acetate filters).
Serial dilutions then were prepared to determine the phage titer.
The phage preparations were stored in SM buffer with a drop of
chloroform at 4.degree. C.
[0070] These bacteriophage preparations then were tested against E.
coli isolates using a ratio of about 1:100 (phage:bacteria) by
infecting about 5 ml aliquot of E. coli with the appropriate amount
of phage, and the tubes were allowed to remain at room temperature
for fifteen minutes. About 1 liter of LB was inoculated, and
incubated overnight at 37.degree. C. with vigorous shaking (250
rpm). In the morning, 10 ml chloroform/l was added and shaking
continued for fifteen minutes. Bacterial debris was pelleted by
centrifugation at 10,000 rpm for fifteen minutes at 4.degree. C.
(Sorvall GSA rotor). Supernatant was poured off into a clean flask,
and the volume was measured. Then, about 5M NaCl (1 ml/100 ml) was
added and mixed by gentle stirring. PEG 8000 (10 g/100 ml) then was
added and allowed to dissolve completely. Phage precipitated
overnight at 4.degree. C. In the morning, the flask was swirled to
resuspend the precipitate, and centrifuged at 5000 rpm for twenty
minutes at 4.degree. C. The supernatant was decanted, and tubes
inverted on a paper towel to dry for several minutes. Pellets then
were resuspended by gentle swirling in a total volume of 5 ml TM
buffer.
[0071] CsCl was added to a density of 1.5 g/ml, and the solution
was placed in ultracentrifuge tubes. Tubes were filled to the neck
with premade CsCl solution (d=1.5 g/ml). Samples were centrifuged
at 34 K overnight at 15.degree. C. (Sorval 865.1 Ti rotor). After
centrifugation, each band was carefully drawn off using an 18 gauge
needle, and placed in a new tube. Each sample was dialyzed
(Spectra-Por Membrane, Molecular Weight Cut-Off 50,000) in 1.5 L
low salt buffer with gentle stirring for about one hour. This step
was repeated twice more, and a titer of about 10.sup.11 was
obtained for each phage. The phage preparations then were stored in
low salt buffer at 4.degree. C.
[0072] Two particularly virulent preparations were selected on the
basis of concentration and isolate sensitivity. CsCl purified
preparations 146A and 173A were capable of killing the majority of
different isolates, and were virulent at a concentration of 100
times less than the least virulent preparation. Purified 146A and
173A phage preparations were tested against 52 different E. coli
isolates, whereby 146A plaqued against 22 isolates (42%) and 173A
plaqued against 20 isolates (38%). Combined, these two phage
preparations were effective against 31 of the 52 isolates (60%). In
comparison, a purified 222A phage preparation was found to be
effective in killing only 6 isolates (11.5%). Purified 146A and
173A E. coli bacteriophage preparations were then characterized by:
(i) plaque morphology (the 146A preparations provided clear plaques
of 1-2 mm with defined edges, and the 173A preparations provided
plaques of 4-6 mm with clear centers of 2-3 mm and turbid haloes);
(ii) electron microscopy (FIGS. 1 and 2); and (iii) DNA
fingerprinting analysis (FIGS. 3 and 4). Samples of virulent
bacteriophage 146A and 173A preparations were deposited in the
ATCC, 12301 Parklawn Drive, Rockville, Md., 20852 on Apr. 15, 1997
and accorded ATCC accession Nos. ______ and ______,
respectively.
Example 7
[0073] Bacterial cultures of Citrobacter fruendii and from
Klebsiella oxytoca were obtained from urinary samples and purified.
Host-specific plaque purified non-toxic bacteriophage preparations
were selected, isolated and purified from sewage filtrate using the
procedures outlined above in Example 6, except that purified
urinary cultures of Citrobacter fruendii and Klebsiella oxytoca
were used. Virulent bacteriophage 262A preparations against C.
fruendii were characterized by: (i) plaque morphology (the C.
fruendii plaques had diameters of 2-4 mm with irregular edges and
distinct centers); (ii) electron microscopy (FIG. 5); and (iii) DNA
fingerprinting analysis. A sample of virulent bacteriophage 262A
preparation was deposited in the ATCC at the above address, on Apr.
15, 1997 and accorded ATCC accession No. ______. Virulent
bacteriophage 174A preparations against K. oxytoca were
characterized similarly by plaque morphology, electron microscopy
and DNA fingerprint analysis (FIG. 6). A sample of virulent
bacteriophage 174A preparation was deposited in the ATCC at the
above address on Apr. 15, 1997 and accorded ATCC accession No.
______.
Example 8
[0074] A number of purified clinical samples of Streptococcus
pyogenes were obtained, some from pus samples, and most from throat
samples. Three phage preparations were obtained from ATCC:
12202-B1, 12203-B1 and 12204-B1. An additional clinical strep phage
was obtained, A25, which appears to be to be identical to ATCC
phage 12204-B1. After preparing A25 in accordance with the present
invention, however, the resulting phage preparation was non-toxic,
highly purified, host-specific, virulent and had a wide host range
whereas the commercially available phage 12204-B1 did not have all
of these characteristics. Bacteriophage preparations were tested
against S. pyogenes isolates as described in Example 6 above, and
the bacteriophage A25 preparation was found to be especially
virulent on the basis of concentration and isolate sensitivity. The
bacteriophage A25 preparations were characterized by plaque
morphology (tiny plaques and circles of complete lysis) and
electron microscopy.
Example 9
[0075] A number of clinical samples of Staphylococcus aureus were
obtained, and purified as described above in example 7. These
samples were isolated from pus samples, throat samples, and nose
swabs. Three phage preparations were obtained from ATCC: ATCC
27706-B1 (equivalent to CDC phage 83A); ATCC 27708-B1; and ATCC
15752-B1 (from nontypeable S. aureus). In addition, two clinical
phage preparations were obtained, 11.phi. and 80.alpha..
[0076] Bacteriophage preparations were tested against S. aureus
isolates as described in Example 6 above, and the bacteriophage 83A
was found to be particularly virulent. Out of nine different host
samples of S. aureus, bacteriophage 83A was effective in killing 5
(56%), whereas the next most virulent bacteriophage after
undergoing the above treatment, was effective in killing only 1
(11%). Thus, 83A bacteriophage had a wider host range than and
killed 400% more bacteria. Bacteriophage obtained directly from the
ATCC, and the clinical samples that did not undergo the above
treatment (i.e., wild-type bacteriophage), were not effective (0%).
The bacteriophage 83A preparations were characterized by plaque
morphology (clear plaques of 1-2 mm with defined edges, electron
microscopy, and DNA fingerprint analysis (FIG. 9).
[0077] While the invention has been described with reference to
particularly preferred embodiments and examples, those skilled in
the art will appreciate that various modifications can be made to
the invention without significantly departing from the spirit and
scope thereof. All of the above-mentioned documents are
incorporated by reference herein in their entirety.
* * * * *