U.S. patent application number 17/544733 was filed with the patent office on 2022-03-31 for methods of identifying bacteriophages that can infect and kill host-adapted pathogenic bacteria.
This patent application is currently assigned to United States of America as Represented by the Secretary of the Navy. The applicant listed for this patent is James M Regeimbal, Stuart D. Tyner. Invention is credited to James M Regeimbal, Stuart D. Tyner.
Application Number | 20220096576 17/544733 |
Document ID | / |
Family ID | 1000006015504 |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220096576 |
Kind Code |
A1 |
Regeimbal; James M ; et
al. |
March 31, 2022 |
Methods of Identifying Bacteriophages that can Infect and Kill
Host-Adapted Pathogenic Bacteria
Abstract
The subject matter of the instant invention relates to methods
of enhancing harvesting of phages against a targeted host bacteria,
as well as methods of identifying phages likely to have an enhanced
propensity to infect and kill an infectious pathogenic bacteria in
vivo, from samples comprising phages. The invention also relates to
phage libraries, pharmaceutical compositions, methods of treatment,
and phage-based diagnostic methods and methods of detecting
bacteria related thereto.
Inventors: |
Regeimbal; James M;
(Washington's Crossing, PA) ; Tyner; Stuart D.;
(Great Falls, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regeimbal; James M
Tyner; Stuart D. |
Washington's Crossing
Great Falls |
PA
VA |
US
US |
|
|
Assignee: |
United States of America as
Represented by the Secretary of the Navy
Silver Spring
MD
United States of America as Represented by the Secretary of the
Army
Silver Spring
MD
|
Family ID: |
1000006015504 |
Appl. No.: |
17/544733 |
Filed: |
December 7, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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16112148 |
Aug 24, 2018 |
11224626 |
|
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17544733 |
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62550461 |
Aug 25, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/76 20130101;
C12N 2795/00032 20130101; C12N 2795/00051 20130101; C12N 7/00
20130101 |
International
Class: |
A61K 35/76 20060101
A61K035/76; C12N 7/00 20060101 C12N007/00 |
Claims
1. A phage library comprising phages harvested against targeted
host bacteria from a sample comprising said phages, said harvesting
method comprising culturing aliquots of said sample in a plurality
of in vitro cultures comprising said targeted host bacteria in
various concentrations of homogenates of mammalian organ, muscle,
and bone.
2. The phage library of claim 1, wherein said culturing step
produces one or more changes in the targeted host bacteria that
occurs in vivo during host-adaptation.
3. A phage library comprising phages with enhanced propensity to
infect and kill an infectious pathogenic bacteria in vivo, wherein
said phages are identified according to a method comprising: a.
culturing the infectious pathogenic bacteria in a plurality of in
vitro cultures comprising various concentrations of homogenates of
mammalian organ, muscle, and bone; b. culturing a sample comprising
phages in said plurality of in vitro cultures from step a; and c.
assaying said plurality of in vitro cultures to identify phages
that can infect and kill the infectious pathogenic bacteria in
vitro in said various concentrations of homogenates of mammalian
organ, muscle and bone.
4. The phage library of claim 2 or claim 3, wherein said culturing
produces changes in expression of one or more genes encoding
bacterial surface features used as phage receptors.
5. The phage library of claim 1 or claim 3, wherein said phage
library is against a MDR bacterial pathogen selected from the group
consisting of Enterococcus faecium, Staphylococcus aureus,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa, and Enterobacter spp.
6. The phage library of claim 1 or claim 3, wherein the sample
comprising phages comprises one or more phages found in nature.
7. The phage library of claim 1 or claim 3, wherein the sample
comprising phages is collected from one or more natural and/or
man-made sources.
8. The phage library of claim 7, wherein said one or more natural
and/or man-made sources is selected from the group consisting of
soil, water treatment plants, raw sewage, sea water, lakes, rivers,
streams, cesspools, animal intestines, human intestines, manure or
other fecal matter, organic substrates, biofilms, and
medical/hospital sources.
9. The phage library of claim 1 or claim 3, wherein the culturing
is under further various conditions selected from the group
consisting of temperature, time, osmotic pressure, pH, CO2
percentage, O2 percentage, nutrient concentration(s), carbon
source(s), carbon source concentration(s), growth factor
concentration(s), hormone concentration(s), in vitro culture
surface characteristics, and concentration of inducer(s) of
bacterial virulence factors.
10. The phage library of claim 9, wherein the nutrients are
selected from the group consisting of amino acids, carbohydrates,
vitamins, and minerals.
11. The phage library of claim 10, wherein the minerals are
selected from the group consisting of iron and magnesium.
12. The phage library of claim 1 or claim 3, wherein the mammalian
organ, muscle, and bone are from a mouse.
13. The phage library of claim 1 or claim 3, wherein the various
concentrations of homogenates of mammalian organ, muscle, and bone
are 5-25% by weight of said plurality of in vitro cultures.
14. The phage library of claim 1 or claim 3, wherein the organ is
selected from the group consisting of liver, brain, heart, spleen,
and kidney.
15. The phage library of claim 1 or claim 3, wherein the plurality
of in vitro cultures further comprise one or more additional
culture additives selected from the group consisting of whole or
fractionated mammalian serum, whole or fractionated mammalian
plasma, and whole mammalian blood.
16. The phage library of claim 15, wherein said whole or
fractionated mammalian serum is selected from the group consisting
of human serum, animal serum, and a combination thereof.
17. The phage library of claim 15, wherein said whole or
fractionated mammalian serum is added to said plurality of in vitro
cultures at a concentration of 0-15%.
18. The phage library of claim 17, wherein the concentration is
7.5%.
19. The phage library of claim 16, wherein the animal serum is
fetal bovine serum (FBS).
20. The phage library of claim 15, wherein said whole or
fractionated mammalian plasma is selected from the group consisting
of human plasma, animal plasma, and a combination thereof.
21. The phage library of claim 15, wherein said whole or
fractionated mammalian plasma is added to said plurality of in
vitro cultures at a concentration of 0-15%.
22. The phage library of claim 21, wherein the concentration is
7.5%.
23. The phage library of claim 15, wherein said whole mammalian
blood is selected from the group consisting of human blood, animal
blood, and a combination thereof.
24. The phage library of claim 15, wherein said whole mammalian
blood is added to said plurality of in vitro cultures at a
concentration of 0-15%.
25. The phage library of claim 24, wherein the concentration is
5%.
26. The phage library of claim 23, wherein the animal blood is
sheep blood.
27. The phage library of claim 15, wherein said fractionated
mammalian serum and said fractionated mammalian plasma may be
fractionated by heat, centrifugation, or biochemically using column
chromatography prior to addition to the plurality of in vitro
cultures.
28. The phage library of claim 15, wherein said whole or
fractionated mammalian serum and said whole or fractionated
mammalian plasma may or may not be heat inactivated.
29. A composition comprising one or more phages of the phage
library of claim 1 or claim 3.
30. A method of treating a bacterial infection in a subject in need
thereof comprising administering to the subject an effective amount
of the composition of claim 29.
31. A method of diagnosing or detecting bacteria in a biotic or
abiotic sample comprising using one or more phages of the phage
library of claim 1 or claim 3.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S.
Non-Provisional patent application Ser. No. 16/112,148 filed Aug.
24, 2018 and which claims the benefit of U.S. Provisional Patent
Application No. 62/550,461 filed Aug. 25, 2017, the entire
disclosures of which are incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The subject matter of the instant invention relates to
methods of enhancing harvesting of bacteriophages ("phages")
against a targeted host bacteria from a sample comprising phages.
Particularly, the methods of the invention are directed to
increasing phage harvesting efficiency as well as enhancing the
yield and diversity of phages isolated from environmental samples.
The focus of the invention also includes improved methods of
isolating phages having an enhanced propensity for infecting and
killing bacterial pathogens in vivo, including host-adapted
infectious pathogenic bacteria. It is contemplated herein that the
methods of the instant invention may be used to create robust
collections of phages ("phage libraries") comprising phages with
greater diversity against bacterial strains, including multidrug
resistant (MDR) bacterial pathogens, than phage libraries prepared
according to conventional methods. It is also contemplated herein
that the methods and compositions of the instant invention will
facilitate not only the design of phage therapeutics with superior
clinical efficacy, but also provide phage-based diagnostic methods
as well as methods of bacterial detection for industrial
applications which provide superior performance.
BACKGROUND OF INVENTION
[0003] MDR bacterial infections are an increasing problem for
military and civilian populations alike. Military populations are
at an especially increased risk for resistant bacterial infections
as traumatic injuries sustained during combat and military service
are highly susceptible to infection, and often require prolonged
hospitalization, further increasing the risk of drug-resistant
nosocomial infections. For example, MDR ESKAPE pathogens
(Enterococcus faecium, Staphylococcus aureus, Klebsiella
pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and
Enterobacter spp.) are known to cause a significant number of the
infectious complications in wounded service members, and these
organisms greatly increase morbidity and mortality. Treatment
options for these kinds of resistant infections are extremely
limited and there is a paucity of new drugs in the pharmaceutical
pipeline.
[0004] In recent years, phage therapy has re-emerged as a potential
alternative treatment for MDR bacterial infections. Lytic phages
(bacterial viruses) are extremely abundant obligate intracellular
parasites that infect, replicate within, and kill very specific
bacterial hosts. Clinically, phage therapy has typically involved
using combinations of different lytic phages deemed likely to
target and kill a specific bacterial pathogen. Recently, advances
in phage therapeutics and antibacterial phage therapy have played a
role in several surprisingly highly successful eIND cases in the
U.S., and in view of promising advances such as these in the field,
there is continued interest in pursuing clinical trials
investigating different therapeutic modalities comprising
phages.
[0005] Notably, phages can be very strain-specific. Thus, in
contrast to early efforts in the field of phage therapeutics, and
as clearly demonstrated in recent clinical eIND cases, phage
therapy may fundamentally require a significant amount of
personalization. That said, whether the phage therapeutic comprises
population level cocktails, engineered phages, or involves a phage
library-to-cocktail personalized therapeutic approach, all of these
modalities would benefit greatly from a coordinated and massive
expansion of phage libraries against bacterial pathogens, including
MDR pathogens such as the ESKAPE pathogens. See, e.g., US
2017/0368116 A1, the entire contents of which are incorporated by
reference herein.
[0006] In addition to clinical uses in antibacterial pharmaceutical
compositions, phage specificity for its target bacterial host may
be exploited clinically in methods of diagnosing bacterial
infections via a phage-based diagnostic. By culturing clinical
samples in the presence of a phage-based diagnostic, the increase
in the phage population would indicate the presence of the phage's
bacterial host. Thus, by monitoring the phage population, the
identification of a specific bacterial pathogen(s) in a clinical
sample can be achieved rapidly without the need for bacterial
strain isolation. Moreover, phage specificity may also be exploited
in methods of detecting bacteria in the environment or in
industrial samples, again through the use of a phage-based
diagnostic. See, e.g., U.S. Ser. No. 15/994,855 the entire contents
of which are incorporated by reference herein. Significantly, since
phages require a viable host in which to replicate, phages can
discriminate between live bacterial cells and the presence of dead
bacterial cells or cell debris, which other molecular detection
technologies cannot manage. Thus, in addition to clinical
applications in therapeutics and diagnostic methods, phages may
also be employed in industrial settings, e.g., where the need to
detect live bacterial contamination is a concern.
[0007] Phage-based methods of detection and diagnosis take
advantage of the ability of a phage to infect and replicate within
its bacterial host such that phage titer often increases 10 to even
100-fold in a single generation, constituting a massive increase in
a specific "signal." This massive increase in the phage titer
"signal" can be exploited for industrial and clinical purposes by
monitoring the increase in phage titer using a number of techniques
comprising, e.g., classical phage titer counts, quantitative
real-time PCR, probes of the phage genome or other reporter
constructs, nucleic acid hybridization or other molecular assays,
fluorescence or immunofluorescence assays with labeled phage
particles, etc.
[0008] While phage-based diagnostics and methods of detection
represent a powerful tool, such methodologies are also constrained
by the same lack of robust collections of phages which currently
restricts the development of phage therapeutics. Indeed, phage
specificity for its bacterial host is both a strength and a
weakness of phage therapeutics; a major hindrance to the continued
advancement of all available phage therapeutic modalities is the
current lack of sufficiently diverse phage libraries which
demonstrate promise for clinical and/or industrial use.
Specifically, robust phage libraries against MDR bacterial
pathogens, and especially against each of the ESKAPE pathogens, are
urgently needed.
[0009] Establishing a diverse and robust phage library for clinical
and/or industrial uses first requires the isolation of appropriate,
relevant phages from the staggering number of phages in the wild.
Traditionally, classical phage harvesting first involves mixing raw
environmental water samples (rich in diverse phages) with culture
media (often in powder form) and a high titer of bacteria against
which phages are desired. Ideally, several different bacterial
strains are provided with the culture media simultaneously (e.g.,
from 1-10 bacterial strains in some cases), and these added
bacterial strains facilitate the expansion of any phage populations
against said bacteria present in the sample. Typically, this mixed
culture is then grown overnight at 37.degree. C. with shaking
(aerobically) to allow for phage expansion and the enrichment of
phages against the said targeted bacteria. The phage-rich
supernatant of this culture is then subjected to classical plaquing
assays to identify whether phages against the targeted bacterial
strain(s) are present in this expanded and mixed phage population.
If such phages are present, they are isolated and purified. This
general method is currently used for phage harvesting and library
construction for both therapeutics and diagnostics.
[0010] Notwithstanding the usefulness of current conventional
methods, the fact remains that classical phage harvesting
methodologies have several limitations. For example, using
currently available harvesting methods, any phages against the
targeted bacterial strains in the mixed phage population of a phage
harvesting culture will emerge based on an in vitro competition
among all the phages present in the environmental sample. As such,
it is contemplated herein that the "winners and losers" of this
interphage competition may reflect, and depend upon, the culture
conditions employed. As a result, there may be valuable phages
present in a conventional harvesting culture that might have
industrial and/or clinical uses against the targeted bacterial
strains, but which get relentlessly out-competed in the milieu of
classical in vitro harvesting culture conditions. Thus, it is
contemplated herein that phage libraries built using only classical
in vitro cultures and growth conditions cannot fully exploit the
vast extent of phage diversity present in the wild.
[0011] While clearly indispensable to biomedical research, one of
skill in the art appreciates the inherent limitations of in vitro
cultures; while extremely useful, data provided from such cultures
may not truly reflect the complexity of in vivo conditions. Thus,
it is further contemplated herein that conventional harvesting
cultures may enrich for phages from the wild that are specific to
the targeted host bacteria when grown in the in vitro physiological
conditions of the microbiological culture media, but not specific
to the same target bacteria under in vivo conditions, i.e., when in
the form of a "host-adapted bacteria" growing in a human or animal
host. Thus, phage libraries for in vivo therapeutic and diagnostic
use built using classical methods may be unfortunately skewed by
comprising phages with limited usefulness for in vivo applications.
Thus, taken together with interphage competition, phages which
demonstrate in vitro specificity for a targeted bacterial strain
not only may prove to have limited clinical use against said
bacteria present under the physiological conditions of the human or
animal host, but also such phages in in vitro harvesting cultures
may actually be out-competing other phages in the culture that
might be better able to infect the same target bacteria when
host-adapted in vivo.
[0012] In view of the foregoing, there remains a need not only for
improved methods of enhancing the isolation of different phages
from the wild, e.g., from environmental samples, but also for
improved methods for identifying phages which can infect and kill
targeted bacterial pathogens, and especially host-adapted
infectious pathogenic bacteria in vivo. Such improved methods would
facilitate the creation of robust phage libraries of enhanced
diversity for use in all types of industrial and clinical
applications, including but not limited to clinical modalities
comprising the use of personalized phage therapeutics with improved
clinical efficacy, as well as for improved methods of detecting
and/or diagnosing bacteria in both clinical and non-clinical
settings via phage-based diagnostics.
SUMMARY OF THE INVENTION
[0013] In a first aspect, the invention relates to a method of
enhancing harvesting of phages against a targeted host bacteria
from a sample comprising phages, said method comprising culturing
aliquots of said sample in a plurality of cultures comprising said
targeted host bacteria, wherein said plurality of cultures
comprises different culture conditions, wherein each of said
culture conditions is designed to support growth of said bacteria
and produce physiological differences in said bacteria in said
plurality of cultures to promote different phage competition
outcomes in said plurality of cultures.
[0014] In another aspect, the invention relates to a method of
identifying phages likely to have an enhanced propensity to infect
and kill an infectious pathogenic bacteria in vivo, said method
comprising:
[0015] a. culturing the infectious pathogenic bacteria in one or
more cultures in vitro, wherein said one or more cultures comprises
culture conditions comprising one or more culture features and/or
additives designed to produce a physiological state and/or gene
expression pattern in said infectious pathogenic bacteria in vitro
that is more similar to that of said infectious pathogenic bacteria
when host-adapted in vivo;
[0016] b. culturing a sample comprising phages in said one or more
cultures; and
[0017] c. assaying said one or more cultures to identify phages in
the sample that can infect and kill the infectious pathogenic
bacteria in vitro under said culture conditions, wherein said
identified phages are likely to have an enhanced propensity to
infect and kill the infectious pathogenic bacteria in vivo.
[0018] In one embodiment of the above aspects, the culture
conditions produce one or more changes in the bacteria that occurs
in vivo during host-adaptation. In one embodiment of the above
aspects, the culture conditions produce one or more changes in gene
expression in the bacteria. In a particular embodiment of the above
aspects, said changes in gene expression comprises changes in genes
expressing bacterial surface features used as phage receptors.
[0019] In various embodiments, the methods of the invention are
used to harvest and/or identify phages from any possible source. In
particular embodiments of the above aspects, the sample comprising
phages comprises one or more wild phages. In another embodiment,
the sample comprising phages comprises one or more previously
isolated phages. In a particular embodiment, the previously
isolated phage is obtained from academic, commercial, or
non-commercial sources.
[0020] In a particular embodiment, the sample comprising phages is
collected from one or more natural or man-made sources. In
particular embodiments, the source is selected from the group
consisting of soil, water treatment plants, raw sewage, sea water,
lakes, rivers, streams, standing cesspools, animal intestines,
human intestines, manure or other fecal matter, organic substrates,
biofilms, and medical/hospital sources.
[0021] In another embodiment, the culture conditions comprise
variations in one or more culture features or additives selected
from the group consisting of culture temperature, culture time,
osmotic pressure, pH, CO.sub.2 percentage, O.sub.2 percentage,
nutrient concentration(s), carbon source(s), carbon source
concentration(s), growth factor concentration(s), hormone
concentration(s), in vitro culture surface characteristics, and
concentration of inducer(s) of bacterial virulence factors. In a
particular embodiment, the nutrients are selected from the group
consisting of amino acids, carbohydrates, vitamins, and minerals.
In a particular embodiment, the variations in culture feature or
additives are selected from the group consisting of iron
concentration, magnesium concentration, concentration of whole or
fractions of mammalian serum, concentration of whole or fractions
of mammalian plasma, concentration of mammalian blood, and
concentration of mammalian tissue homogenates, including
homogenates of organs, muscle, and bone. In a particular
embodiment, the mammalian serum is fetal bovine serum (FBS). In a
particular embodiment, the additive is sheep blood. In a particular
embodiment, the additive may or may not be a heat-inactivated
substance.
[0022] In a particular embodiment, the culture conditions and/or
the culture surface characteristics support the growth of the
targeted host bacteria in a biofilm. In a particular embodiment,
said culture surface comprises materials selected from the group
consisting of plastics, metals, surfaces coated with complex host
extracts, tissue lysates, biological homogenates, cells, cells
debris, and bone.
[0023] In one embodiment, the culture surface comprises materials
selected from the group consisting of biotic and abiotic surfaces.
In a particular embodiment the biotic surface comprises materials
selected from the group consisting of collagen, bone, tissue
explants, tissue lysates, homogenized tissue material, human or
mammalian cells, and cell debris. In another particular embodiment,
the abiotic surface comprises materials selected from the group
consisting of metals and plastic. In a particular embodiment, the
metals are selected from the group consisting of stainless steel,
titanium, and aluminum.
[0024] In a particular embodiment, the culture temperature is less
than about 37.degree. C. In another embodiment, the culture
temperature is greater than about 37.degree. C. In yet another
embodiment, the culture temperature is about 10.degree. C. to about
42.degree. C. In another embodiment, the culture temperature is
about 20.degree. C. to about 25.degree. C. ("room
temperature").
[0025] In another particular embodiment, the iron concentration is
about 0 .mu.M to less than about 0.3 .mu.M. In another embodiment
the iron concentration is less than about 2 .mu.M. In a particular
embodiment, the iron provided is selected from the group consisting
of chelated-iron, nitrate salts, and sulfate salts. In a particular
embodiment, the iron concentration is a limiting iron
concentration.
[0026] In yet another particular embodiment, the serum
concentration is about 0-15%. In a particular embodiment, the serum
concentration is about 7.5%. In another embodiment, the serum is
selected from the group consisting of human serum, animal serum,
and a combination thereof. In a particular embodiment, the animal
serum is fetal bovine serum (FBS). In a particular embodiment, the
serum is provided as whole serum. In a particular embodiment, the
serum may be fractionated by heat, centrifugation, or fractioned
biochemically using column chromatography prior to addition to the
cultures. In a particular embodiment, the serum may or may not be
heat inactivated. In a particular embodiment, the cultures comprise
7.5% FBS.
[0027] In yet another particular embodiment, the plasma
concentration is about 0-15%. In a particular embodiment, the
plasma concentration is about 7.5%. In another embodiment, the
plasma is selected from the group consisting of human plasma,
animal plasma, and a combination thereof. In a particular
embodiment, the plasma is provided as whole plasma. In a particular
embodiment, the plasma may be fractionated by heat, centrifugation,
or biochemically using column chromatography prior to addition to
the cultures. In a particular embodiment, the plasma may or may not
be heat inactivated.
[0028] In yet another particular embodiment, the whole blood
concentration is about 0-15%. In yet another particular embodiment,
the whole blood concentration is about 5%. In a particular
embodiment, the whole blood is selected from the group consisting
of human blood, animal blood, and a combination thereof. In a
particular embodiment, the animal blood is sheep blood. In a
particular embodiment, the cultures comprise 5% sheep blood.
[0029] In another embodiment, the concentration of inducer(s) of
bacterial virulence factors is about 0-15%. In a particular
embodiment, the inducer(s) of bacterial virulence factors is
selected from the group consisting of charcoal,
glucose-6-phosphate, cholesterol, and fetal bovine serum (FBS). In
a particular embodiment, the concentration of glucose-6-phosphate
or cholesterol is about 2 g/L and about 100 mg/L, respectively.
[0030] In a particular embodiment, the nutrient concentration is
limited by using minimal media in the cultures for bacterial
growth. In another embodiment, the pH is from about pH 6.5 to about
pH 8.5.
[0031] In yet another embodiment, the CO.sub.2 percentage is about
0-7% CO.sub.2. In a particular embodiment, the CO.sub.2 percentage
is about 5% CO.sub.2.
[0032] In yet another embodiment, the O.sub.2 percentage in about
0-20% O.sub.2. In a particular embodiment, the O.sub.2 percentage
is less than about 2% O.sub.2.
[0033] In another embodiment, the cultures may be grown for less
than about 18 hours. In another embodiment, the cultures are grown
about 8 hours. In another embodiment, the cultures are grown
overnight. In another embodiment, the cultures may be grown for
about 18-36 hours or more.
[0034] In another embodiment, the cultures are grown aerobically or
non-aerobically (with or without shaking the cultures), or a
combination of both. In a particular embodiment, the cultures are
grown with shaking at 250 rpm.
[0035] In another aspect, the invention relates to a composition
comprising one or more phages identified according to the methods
of the instant invention. In a particular embodiment, the
composition is a pharmaceutical composition. In another embodiment,
the composition is for use with diagnostic methods or methods of
detecting bacteria.
[0036] In yet another aspect, the invention relates to a phage
library comprising one or more phages identified according to the
methods of the instant invention. In one embodiment, the phage
library is against a MDR bacterial pathogen. In a particular
embodiment, the phage library is against an MDR ESKAPE bacterial
pathogen selected from the group consisting of Enterococcus
faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter
spp.
[0037] In yet another aspect, the invention relates to a method of
treating a bacterial infection in a subject in need thereof
comprising administering to the subject an effective amount of a
pharmaceutical composition comprising one or more phages identified
according to the methods of the instant invention. In a particular
embodiment, the bacterial infection to be treated is selected from
the group consisting of wound infections, surgical hardware
associated infections, post-surgical infections, and systemic
bacteremia.
[0038] In additional aspects, the invention relates to methods of
diagnosing or detecting bacteria in clinical and non-clinical
settings comprising employing phages harvested according to the
methods of the instant invention and/or derivatives thereof, e.g.,
engineered phages. In particular embodiments, the methods of the
instant invention are used to diagnose or detect bacteria in biotic
and/or abiotic samples. In a particular embodiment, the biotic
sample is a sample from an infected mammalian host. In a particular
embodiment, the biotic sample is selected from the group consisting
of blood samples, sputum samples, swabs from mucus membranes or
wounds, biopsies, and puss. In a particular embodiment, the abiotic
sample is selected from the group consisting of industrial samples,
food samples, pharmaceuticals, makeup and beauty products, and
swabs from machinery.
[0039] In a particular embodiment, the invention relates to methods
of detecting or diagnosing the presence of a targeted bacteria,
said method comprising harvesting phages according to the methods
of the instant invention, and employing one or more of said
harvested phages in assays to diagnose or detect said targeted
bacteria. In a particular embodiment, said assays comprise
detecting levels of phage infection and/or phage titer levels,
wherein evidence of phage infection and/or an increase in phage
titer levels indicate presence of the targeted bacteria. In
particular embodiments, the phage titer levels are assayed using
methods selected from the group consisting of plaquing assays, PCR,
nucleic acid hybridization, labeling, and immunolabeling. In a
particular embodiment, the PCR method is real-time PCR.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 depicts host ranges of various A. baumannii (AB)
phages isolated from the Peruvian Amazon in 2017. The magnitude of
the bar is the total number of AB strains tested that each phage
can infect.
[0041] FIG. 2 depicts the host ranges of various K. pneumoniae (KP)
phages isolated from the Peruvian Amazon in 2017. The symbol *
indicates phages tested against only ten strains of KP, not twenty
strains of KP. The different color bars represent different phages
isolated against the same strain on the X axis. The magnitude of
the bar is the total number of KP strains tested that each phage
can infect.
[0042] FIG. 3 depicts the host ranges of various P. aeniginosa (PA)
phages isolated from the Peruvian Amazon in 2017. The different
color bars represent different phages isolated against the same
strain on the X axis. The magnitude of the bar is the total number
of PA strains tested that each phage can infect.
[0043] FIG. 4 depicts the host ranges of various S. aureus (SA)
phages isolated from the Peruvian Amazon in 2017. The magnitude of
the bar is the total number of SA strains tested that each phage
can infect.
[0044] FIG. 5 depicts data collected from K. pneumoniae (KP) phages
harvested via traditional and conditional cultures. Each row is
specific for a K. pneumoniae host strain, and each phage image
(hexagon) represents an isolated phage under each condition
(column). Per row, the color/shading of the phage represents host
range, with the same color indicating the phages had identical
host-ranges among the complete set of 20 K. pneumoniae strains
tested. The number inside of the phage figure (hexagon) is the
number of strains that each phage infects, including its host. The
"+" means the yield of phages was significantly increased in that
condition.
[0045] FIG. 6, FIG. 7, FIG. 8A, FIG. 8B, FIG. 9A, FIG. 9B, FIG.
10A, and FIG. 10B depict data for studies described in Example 2.
FBS, 7.5% fetal bovine serum; SB, 5% sheep blood; RT, room
temperature; Sh, with shaking (250 rpm); non-SH, without shaking.
Each type of hexagon (solid, dashed, dotted, colored) represents a
different phage that was isolated against the host strain in
question (row) and under the condition in question (column), as
defined by host range against a set of 20 different host strains of
the same species in question (K. pneumoniae in FIGS. 6-7, P.
aeruginosa in FIGS. 8A, 8B, 9A and 9B, and A. baumannii in FIGS.
10A and 10B.) The three ratios of numbers in the hexagon indicate
the following: The top ratio refers to the number of strains that
the phage infected over 20 strains on plates with only tryptic soy
broth (TSB); the middle ratio refers to the number of strains that
the phage infected over 20 strains on plates in TSB with FBS added;
the bottom ratio refers to the number of strains that the phage
infected over 20 strains on plates in TSB with SB added. Each row
is independent, the same outline pattern/color in each row means
that the phage infected the same strains under the same conditions.
Different color in each row means that the phages infected
different strains and/or under different conditions.
[0046] FIG. 6 depicts phages that were isolated against the listed
5 MDR strains of K. pneumoniae (KP) using the strategy of the
instant invention in which a single environmental sample was
aliquoted across a set of three harvesting cultures containing: the
listed KP strains, the aliquoted water, TSB, or TSB+7.5% FBS, or
TSB+5% SB.
[0047] FIG. 7 depicts phages that were isolated against the listed
5 MDR strains of K. pneumoniae (KP) using the strategy of the
instant invention in which a single environmental sample was
aliquoted across a set of three harvesting cultures containing: the
listed KP strains, the aliquoted water, TSB, or TSB+7.5% FBS, or
TSB+5% SB.
[0048] FIG. 8A depicts phages that were isolated against the listed
5 MDR strains of P. aeruginosa (PA) using the strategy of the
instant invention in which a single environmental sample was
aliquoted across a set of three harvesting cultures containing: the
listed PA strains, the aliquoted water, TSB, or TSB+7.5% FBS, or
TSB+5% SB.
[0049] FIG. 8B depicts phages that were isolated against the listed
5 MDR strains of P. aeruginosa (PA) using the strategy of the
instant invention in which a single environmental sample was
aliquoted across a set of three harvesting cultures containing: the
listed PA strains, the aliquoted water, TSB, or TSB+7.5% FBS, or
TSB+5% SB. 0/20 ratio for NSI0978 and NSI1485 indicates phages
isolated with SB did not grow in any of the 20 strains tested
including their host original strain.
[0050] FIG. 9A depicts phages that were isolated against the listed
5 MDR strains of P. aeruginosa (PA) using the strategy of the
instant invention in which a single environmental sample was
aliquoted across a set of three harvesting cultures containing: the
listed PA strains, the aliquoted water, TSB, or TSB+7.5% FBS, or
TSB+5% SB.
[0051] FIG. 9B depicts phages that were isolated against the listed
5 MDR strains of P. aeniginosa (PA) using the strategy of the
instant invention in which a single environmental sample was
aliquoted across a set of three harvesting cultures containing: the
listed PA strains, the aliquoted water, TSB, or TSB+7.5% FBS, or
TSB+5% SB.
[0052] FIG. 10A depicts phages that were isolated against the
listed 5 MDR strains of A. baumannii (AB) using the strategy of the
instant invention in which a single environmental sample was
aliquoted across a set of three harvesting cultures containing: the
listed PA strains, the aliquoted water, TSB, or TSB+7.5% FBS, or
TSB+5% SB.
[0053] FIG. 10B depicts phages that were isolated against the
listed 5 MDR strains of A. baumannii (AB) using the strategy of the
instant invention in which a single environmental sample was
aliquoted across a set of three harvesting cultures containing: the
listed PA strains, the aliquoted water, TSB, or TSB+7.5% FBS, or
TSB+5% SB.
[0054] FIG. 11 depicts a possible strategy disclosed herein for a
method of enhancing harvesting of phages against a targeted host
bacteria from a sample comprising phages. The sample can be from
any source. As depicted therein, Steps 1-5 depict creating
different cultures comprising different aliquots of the same
environmental water sample cultured in the presence of the same
bacterial host strain under different culture conditions. As
depicted therein, step 6 comprises collecting phage supernatants
for phage isolation, e.g., using conventional methods or high
throughput. Each culture supernatant obtained in step 6 may contain
differentially enriched subsets of the available phages in the
original environmental sample (1). Without being bound by any
particular theory, it is contemplated herein that these phages may
use different receptors and/or show differing abilities to infect
conditionally adapted bacterial cells (including host-adapted
cells) and/or show conditional dependence for infection and/or show
different tolerances to changing conditions and/or have different
intrinsic characteristics. Thus the methods of the instant
invention can be used to identify phages that may be better (or
worse) for use in therapeutics or diagnostics.
DETAILED DESCRIPTION
[0055] While the specification concludes with the claims
particularly pointing out and distinctly claiming the invention, it
is believed that the present invention will be better understood
from the following description.
[0056] All percentages and ratios used herein are by weight of the
total composition unless otherwise indicated herein. All
temperatures are in degrees Celsius unless specified otherwise. All
measurements made are at 25.degree. C. and normal pressure unless
otherwise designated.
[0057] The present invention can "comprise" (open ended) or
"consist essentially of" the components of the present invention as
well as other ingredients or elements described herein. As used
herein, "comprising" means the elements recited, or their
equivalent in structure or function, plus any other element or
elements which are not recited. The terms "having" and "including"
are also to be construed as open ended unless the context suggests
otherwise. As used herein, "consisting essentially of" means that
the invention may include components in addition to those recited
in the claim, but only if the additional components do not
materially alter the basic and novel characteristics of the claimed
invention.
[0058] All ranges recited herein include the endpoints, including
those that recite a range "between" two values. Terms such as
"about," "generally," "substantially," "approximately" and the like
are to be construed as modifying a term or value such that it is
not an absolute, but does not read on the prior art. Such terms
will be defined by the circumstances and the terms that they modify
as those terms are understood by those of skill in the art. This
includes, at very least, the degree of expected experimental error,
technique error and instrument error for a given technique used to
measure a value. Unless otherwise indicated, as used herein, "a"
and "an" include the plural, such that, e.g., "a phage" can mean at
least one phage, as well as a plurality of phages, i.e., more than
one phage.
[0059] Where used herein, the term "and/or" when used in a list of
two or more items means that any one of the listed characteristics
can be present, or any combination of two or more of the listed
characteristics can be present. For example, if a composition of
the instant invention is described as containing characteristics A,
B, and/or C, the composition can contain A feature alone; B alone;
C alone; A and B in combination; A and C in combination; B and C in
combination; or A, B, and C in combination. The entire teachings of
any patents, patent applications or other publications referred to
herein are incorporated by reference herein as if fully set forth
herein.
[0060] Lytic phages typically infect bacteria by docking to
receptors on the bacterial surface, injecting their genetic
material (usually DNA), producing their progeny within the
bacterial cell using bacterial machinery, and finally lysing the
bacterial cell upon progeny release, killing the bacterial cell in
the process. All of these steps are necessary in order for a phage
to successfully replicate and kill an infected bacterial cell, and
all of these steps are absolutely dependent on the physiological
state and health of said bacterial cell.
[0061] The mechanisms of action regarding phage infectivity and
replication within its target bacterial host poses a challenge in
the design of phage-based therapeutics as well as phage-based
methods of diagnosing or detecting bacteria. As discussed above, it
is well known that in vitro cultures cannot completely duplicate in
vivo conditions. This is true for bacterial cultures, including
cultures of human pathogens such as the ESKAPE pathogens. Thus, a
pathogenic bacterium growing outside of a host very likely does not
express the same proteome as that expressed by said bacterium when
infecting a host, i.e., when the bacterium's gene expression is
"host-adapted." Thus, notably, during phage isolation in vitro, the
targeted bacterial pathogen typically adapts its physiology, gene
expression, and therefore its surface receptor repertoire, in
response to the particular culture conditions used during phage
isolation; i.e., typically, standard growth media grown aerobically
at 37.degree. C. Accordingly, during phage isolation procedures,
all of the phage replication steps that take place within the
bacterial cell in vitro reflect, and thus are naturally limited by,
the parameters of bacterial gene expression set by the in vitro
culture conditions.
[0062] Thus, it is contemplated herein that since standard
classical culture techniques using standard growth media and
temperature may restrict the gene expression pattern of a bacterium
and may result in a receptor repertoire or physiological state that
is unique and/or significantly different from that of the same
bacterium growing in a host in vivo, current in vitro methods of
isolating phages for possible clinical and industrial use may fail
to maximize the isolation of available phages in an environmental
sample, and thus may fail to identify all potentially useful phages
in the sample. Moreover, conventional phage harvesting methods may
in fact actually enrich for phages optimally suited to infecting
and killing bacteria grown in conventional culture conditions, but
not bacteria growing in and causing an infection in a host
organism. As discussed above, bacteria grown under specific culture
conditions may not express surface receptors that are expressed in
other conditions or in vivo, and similarly bacteria in vitro may
express surface receptors that are not expressed at all in vivo.
Without being bound to any particular theory, it is contemplated
herein that such receptors may act as "decoys" in conventional
methods of phage harvesting by selecting for phages that can infect
and kill a bacterial strain in vitro, and/or only under certain
specific or narrow conditions in vitro, but which will actually
have little therapeutic efficacy on the same bacterial strain in
vivo. Thus, phages isolated on bacteria raised in conventional
cultures may be suboptimal or even incapable of infecting
pathogenic bacteria during a bona-fide infection in a human or
animal.
[0063] Accordingly, it is contemplated herein that there remains a
need not only for improved methods of harvesting wild phages from
both man-made and natural sources, but also for identifying phages
which can specifically infect and kill bacterial pathogens in vivo,
especially host-adapted infectious pathogenic bacteria, and
particularly, MDR bacteria. Improved methods for harvesting and
identifying such phages will enable the creation of robust diverse
phage libraries which in turn can be used to create various types
of phage-based therapeutics and diagnostics for clinical
applications. In a particular embodiment, it is contemplated herein
that the methods of the instant invention can be used to develop
diverse phage libraries for compounding personalized phage
therapeutics with improved clinical efficacy.
[0064] Similarly, as discussed above, one of skill in the art will
appreciate that phage diagnostics, though powerful, are limited by
the same constraints as phage therapeutics; detecting bacterial
hosts in biotic or infected host-derived samples may also be
effected by the same gene expression changes in vitro as those
effecting phage therapeutics. Similar concerns limit the
development and use of phage-based methods of detecting bacteria in
industrial applications. Accordingly, isolating phages in vitro
using classical conditions, and not accounting for conditional gene
expression changes in the targeted bacterial host, may produce
phage-based diagnostics and methods of detecting bacteria of
limited value in actual application. Instead, effective phage-based
diagnostics and phage-based detection methods must consider
conditional changes in bacterial gene expression in order to ensure
the fidelity of phage-based products and methods. Varying the
conditions under which phages are harvested for such uses may allow
for the creation of diagnostics and methods of detection that can
function under multiple bacterial physiological and gene expression
states, including those states in vivo.
[0065] It is theorized herein that in a mixed bacterial culture
with a mixed phage population such as that found in a classical
phage harvesting culture, there exists competition between and
among phages for access to mutual host bacteria. Thus, it is
contemplated herein that exploiting this interphage competition in
a harvesting culture, and noting the marked differences that can
occur between the gene expression patterns of pathogenic bacteria
growing inside and outside a host, offers an intriguing means of
optimizing harvesting conditions to favor the isolation of phages
against targeted host-adapted bacteria, by inducing a host-adapted
like state in vitro in phage harvesting cultures. Additionally,
simply varying specific culture conditions across a set of
otherwise identical phage harvesting cultures, may produce relative
bacterial gene expression differences across the set of cultures
and/or modulate interphage competition outcomes, increasing the
diversity of phages harvested across that set.
[0066] Accordingly, the methods of the instant invention are
directed to varying discrete conditional parameters across a set of
otherwise identical in vitro phage harvesting cultures, thereby
modifying the outcome of phage competition across the set, e.g. the
identity of the winning phages (those that increase in titer) and
losing phages (those that fail to produce a detectable increase in
titer), and therefore increasing the diversity of phages which are
eventually isolated from said cultures, e.g., by altering the
physiological state and/or concomitant gene expression of the
bacterial host(s) in each culture. In a particular embodiment, it
is contemplated herein that aliquoting a single sample comprising
phages into a set (plurality) of cultures comprising the same
bacterial target(s) but comprising differing varying physiological
conditions, including culture conditions designed to more closely
mirror growth conditions in the host during a bacterial infection
in vivo, will increase phage isolation diversity and efficiency
across the set and allow for the isolation of phages that can more
reliably infect bacteria growing with a "host-adapted" gene
expression pattern and surface-feature repertoire than phages
isolated according to conventional methods. As a result, the
methods of the instant invention will allow different phages to win
the competition under those different conditions, thereby
increasing the number and diversity of phages isolated from a
single sample, thus enabling the creation of more expansive and
diverse phage libraries.
[0067] Thus, it is contemplated herein that the effective
development of numerous phage-based therapeutic modalities,
including therapeutics comprising natural phage products, phages
engineered to carry antimicrobial cargo, or that possess expanded
host-range, phage-based diagnostics, and methods of detecting
bacterial contamination, would all benefit from the use of phages
that are isolated from diverse culture conditions that can enhance,
and even maximize, the isolation of phages from experimental
samples.
[0068] Accordingly, in a first aspect, the invention relates to a
method of enhancing harvesting of phages against a targeted host
bacteria from a sample comprising phages, said method comprising
culturing aliquots of said sample in a plurality of cultures
comprising said targeted host bacteria, wherein said plurality of
cultures comprises different culture conditions, wherein each of
said culture conditions is designed to support growth of said
bacteria and produce physiological differences in said bacteria in
said plurality of cultures to promote different phage competition
outcomes in said plurality of cultures.
[0069] In another aspect, the invention relates to a method of
identifying phages likely to have an enhanced propensity to infect
and kill an infectious pathogenic bacteria in vivo, said method
comprising:
[0070] a. culturing the infectious pathogenic bacteria in one or
more cultures in vitro, wherein said one or more cultures comprises
culture conditions comprising one or more culture features and/or
additives designed to produce a physiological state and/or gene
expression pattern in said infectious pathogenic bacteria in vitro
that is more similar to that of said infectious pathogenic bacteria
when host-adapted in vivo;
[0071] b. culturing a sample comprising phages in said one or more
cultures; and
[0072] c. assaying said one or more cultures to identify phages in
the sample that can infect and kill the infectious pathogenic
bacteria in vitro under said culture conditions, wherein said
identified phages are likely to have an enhanced propensity to
infect and kill the infectious pathogenic bacteria in vivo.
[0073] As understood herein, a "targeted host bacteria", "target
bacteria" and like terms refers to the bacteria against which one
or more phages are sought. These can include, e.g., infectious,
pathogenic bacteria. Infectious, pathogenic bacteria are familiar
to one of skill in the art and include, but are not limited to, the
MDR bacteria discussed in detail herein.
[0074] While it is contemplated herein that varying amounts of a
sample comprising phages may be used in the methods of the instant
invention, in a particular embodiment, sample aliquots of the same
volume are used in the methods of the instant invention. In a
particular embodiment, constant and discrete amounts of a fluidic
and/or solubilized homogenous sample comprising phages are added
across a plurality or set of liquid cultures of uniform volume all
comprising the same one or more bacterial strains, including target
bacterial strains. In a particular embodiment, solid sources of
phages (e.g. soil, feces, etc.) are first solubilized in water or
other liquid to facilitate aliquoting.
[0075] As described herein, the methods of the instant invention
allow for the increase in phage isolation from a single sample by
aliquoting said sample across a set of phage harvesting cultures,
and varying specific culture conditions across the set of otherwise
identical cultures. Without intending to be limited to any
particular theory or mechanism of action, it is contemplated herein
that the methods disclosed herein produce relative bacterial gene
expression differences, and/or bacterial physiology differences,
and/or modulate interphage competition outcomes across the set of
cultures, increasing the diversity of phages harvested across that
set from the single sample (see, e.g., FIG. 11 which provides a
sample schematic of this concept.) Varying the culture conditions
so as to mimic the host environment, and/or to modulate bacterial
gene expression and/or physiology so as to recapitulate a
"host-adapted" state in the targeted bacteria, may allow for the
isolation of phages better able to infect bacterial pathogens in
vivo, and/or select against phages that infect in vitro adapted
bacteria.
[0076] The ability of the methods disclosed herein to produced
enhanced phage harvesting results is demonstrated in the results
provided in the below examples. For example, FIGS. 6, 7, 8A, 8B,
9A, 9B, 10A and 10B illustrate in numerous bacterial strains and
species that the addition of FBS or sheep's blood to harvesting
cultures not only diversifies phage harvesting across the set of
harvesting cultures, but also that there are phages that are
apparently "conditionally isolated" (when scored by host range,
arguably the most important feature of a phage with respect to its
therapeutic potential). That is, there are phages present in a
single sample that can be isolated in the presence of FBS or
sheep's blood that cannot be found in media-only traditional
cultures, and vice versa. These results illustrate that although
these phages are present across the set of cultures via the
homogenous aliquoted sample, the conditional cultures produce
different "winners" and "losers" from each culture's interphage
competition. These experimental cultures and results directly
illustrate that it is possible to create culture conditions
designed to support growth of bacteria but which also produce
physiological difference in the bacteria that can promote different
phage competition outcomes in the cultures. To this end, as
discussed below in Example 3, future experiments using concentrated
mouse organ homogenates that comprise far more complex host
materials may improve the ability of conditional culture sets to
identify even more phages for clinical and industrial use.
[0077] Given the difference in phage harvesting results described
herein, employing differences in culture features and additives
according to the methods of the instant invention can clearly
promote changes in the physiological state of the cultured bacteria
and thus enhance phage harvesting. As illustrated in the examples
and data provided herein, the method works in multiple different
species, however, the exact mechanism as to how these differences
in culture features and/or additives change the physiological state
of the bacteria, and thus modulate the results of phage
competition, is not known.
[0078] While the mechanism of action in each bacterial species may
vary, the method is nevertheless very robust and likely involves
bacterial gene expression differences across the culture set,
including, e.g., changes in the expression of bacterial surface
proteins that can serve as phage receptors. Thus, in view of these
data, and as discussed below in detail, it is contemplated herein
that the invention includes modifying the culture conditions to
more closely approach in vivo conditions, thus producing a
physiological state and/or gene expression pattern in infectious
pathogenic bacteria in vitro that is more similar to that of said
infectious pathogenic bacteria when host-adapted in vivo.
Accordingly, it is contemplated herein that that such modified
culture conditions will be able to identify phages that are more
likely to have an enhanced propensity to infect and kill an
infectious pathogenic bacteria in vivo.
[0079] It is understood herein that varying bacterial culture
conditions in vitro according to the methods of the instant
invention may not produce bacterial cultures which completely mimic
physiological conditions in vivo. Regardless, even if such modified
cultures can never fully simulate the host environment, it is
contemplated herein that such modifications may nonetheless produce
different and distinct physiological and gene expression states in
bacteria that may be useful to achieve an enhancement in the
isolation of diverse phages from an environmental source compared
to conventional methods of phage harvesting. Notably, undue
experimentation is not necessary to perform the methods described
herein. Indeed, as demonstrated in Example 1 and Example 2 herein,
even a modification of a single culture feature or additive
(+/-FBS) may be useful to enhance the isolation of phages having
different host ranges from a single common water source.
[0080] As discussed herein, the methods of the instant invention
demonstrate that not only are there phages that show conditionally
dependent isolation, but there are phages that show conditionally
independent and conditionally dependent infection of the same host.
For example, as described in Example 2 and depicted in FIG. 6,
harvesting data with bacterial strain WIQ0239 illustrates that a
phage was found against this strain in the presence of sheep's
blood, and that this phage cannot infect the WIQ0239 host without
sheep's blood present. Also, FIG. 6 harvesting data with bacterial
strain WIQ0289 illustrates that there was a phage found that was
capable of infecting the WIQ0289 host in traditional culture media
and under traditional conditions, but cannot infect when either FBS
or sheep's blood is added to the culture. These remarkable results
indicate that there are phages in the wild that cannot infect their
host strain in the presence of biological materials like FBS and
sheep's blood, and there are some phages that are incapable of
infecting their host strains in the absence of such biological
materials. We contemplate herein that it is highly likely these
kinds of phages, both conditionally isolated with and conditionally
dependent on host biological materials, will show marked
differences in their therapeutic utility. Significantly, based on
these data, it is contemplated herein that phages which show a
preference to infect a bacterial host when in the presence of host
biological materials will likely have superior therapeutic
potential and an enhanced propensity to kill host-adapted bacteria
in vivo.
[0081] As understood herein, "enhancing" the harvesting of phages
against a target host bacteria includes but is not limited to
improving the total yield and/or the efficiency of phage isolation
from a sample as compared to conventional methods. It is
contemplated herein that "enhancing" the isolation of phages
according to the methods of the instant invention comprises the
ability to identify a variety of different phages from the same
sample, e.g., more diverse phages may be identified from the same
sample by altering bacterial culture conditions as described in
detail herein. Significantly, it is contemplated herein that the
methods can be used to identify phages having different host
ranges.
[0082] Similarly, phages with "enhanced propensity" to infect and
kill an infectious pathogenic bacteria in vivo identified according
to the methods of the instant invention refers to phages that are
more likely to infect and kill an infectious pathogenic bacteria in
vivo in comparison with phages identified using conventional in
vitro methods.
[0083] As understood herein, "host-adapted" bacteria are bacteria
that have infected an organism, and have undergone a gene
expression change or other modification which facilitates the
ability of the bacteria to grow in the host organism. One of skill
in the art will appreciate that bacterial host adaptation may be
characterized using conventional methods without undue
experimentation, including but not limited to, analyzing and
comparing in vitro and in vivo patterns of gene expression in
infectious pathogenic bacteria.
[0084] Infectious bacteria frequently grow as a biofilm during an
infection. Biofilms are known to have drastically altered cellular
surface features and phage receptors relative to cells grown
planktonically and/or in traditional cultures. Thus, it is further
contemplated herein that the methods of the invention comprise
using cultures of bacteria in biofilms to enhance phage isolation
and/or identify phages with enhanced propensity to infect and kill
infectious pathogenic bacteria in vivo. Accordingly, by varying the
physiological conditions of phage isolation cultures and/or
mimicking in vivo conditions in vitro with conditional cultures
and/or by first growing bacteria as a biofilm and isolating phages
against said biofilm, the phages isolated on bacteria grown in
these varied cultures according to the methods of the instant
invention will maximize diverse phage isolation, for example by
enriching for the use of receptors that are specifically expressed
during bona-fide infections, thus allowing for better formulation
of phage therapeutics and/or diagnostics that more specifically
target host-adapted bacterial pathogens or bacteria growing under
different conditions.
[0085] Conditional Cultures
[0086] As one of skill in the art will appreciate, bacteria may be
grown, or "cultured" in vitro according to a variety of
conventional methods. These conventional cultures include the use
of plates and flasks which provide physical surface areas suitable
for bacterial growth, incubators which provide proper temperature,
humidity, and aerobic or anaerobic conditions, and solid or liquid
culture media which provides necessary chemicals and other reagents
to support bacterial growth. These media may be modified to include
a variety of minerals, nutrients, energy sources, and buffering
agents depending on the bacteria being cultured. It is contemplated
herein that the methods of the present invention comprise modifying
such conventional culture conditions to create various different
"conditional cultures", including various different culture
conditions, wherein each of said culture conditions is designed to
support growth of said bacteria and produce physiological
differences in said bacteria in a plurality of cultures to promote
different phage competition outcomes in the plurality of cultures.
For example, as discussed herein in detail, one or more culture
features and/or culture media additives may be varied to produce a
myriad of culture conditions for use in the methods of the instant
invention.
[0087] Specifically, it is contemplated herein that the conditional
cultures of the instant invention may be designed to mimic
different physiological conditions which occur during bacterial
infection in vivo in order to trigger physiological changes in
pathogenic bacteria that are likely to mirror the bacterial
physiology and gene expression patterns seen during mammalian,
e.g., human, infections. In a particular embodiment, it is
contemplated herein that changing the physiological conditions of
the host bacteria to conditions that better mimic the bacterial
physiology and gene expression during an infection will allow for
the isolation of phages that use a different repertoire of surface
receptors than those available on classical aerobically growing
37.degree. C. cultures. Specifically, it is contemplated herein
that in certain embodiments, culture conditions employed in the
methods of the instant invention support isolation and
identification of phages that recognize a repertoire of surface
receptors better-suited to infecting bacteria growing in a human
host. Modifying culture conditions and thus altering the gene
expression and phage receptor repertoire of the targeted host
bacteria will likely enhance and may even maximize the isolation of
phages present in an environmental sample, leading to a more
diverse phage library from which to build phage-based therapeutics
of all modalities and/or phage-based diagnostics.
[0088] In a particular embodiment, it is contemplated herein that
the use of different culture conditions will trigger different
and/or unique but overlapping bacterial gene expression patterns in
each condition. Importantly, it is believed that some of the gene
expression pattern changes that will occur will be in genes
expressing bacterial surface features used as phage receptors.
Thus, aliquoting a single sample and culturing the same host
strain(s) in a plurality of conditional cultures each containing an
aliquot of said sample, will create a set of cultures comprising
the same bacteria but expressing different phage receptors across
the culture set, allowing each aliquoted conditional culture to
select a different subset of the available phages in said
environmental source for harvesting, and thus an enhanced variety
of different phages may be isolated from a single environmental
source.
[0089] Similarly, it is also contemplated herein that in a
particular embodiment, the culture conditions may be varied to
trigger an expression pattern of one or more genes in an infectious
pathogenic bacteria in vitro that is similar to an expression
pattern of these one or more genes in the infectious pathogenic
bacteria during infection of said host in vivo. It is also
contemplated herein that some of the induced gene expression
pattern changes will be in genes expressing bacterial surface
features used as phage receptors.
[0090] Specific types of conventional media are familiar to one of
skill in the art, and are available from a variety of commercial
vendors. These include, for example, simple or basal media, complex
media, defined media and special media. In various embodiments,
culture media for use in the methods of the instant invention
include commercially available "ready to use" microbial culture
media. Suitable commercially available microbial culture media that
may be used include, e.g., LB. TSB/A, defined rich media, and
defined minimal media (discussed below). According to the methods
of the instant invention, the microbial culture media may be
modified to further comprise various additives and/or to lack
certain reagents by design to vary bacterial gene expression and/or
induce infection-like gene expression in the bacterial pathogen.
Media, reagents, and other additives for use in the methods of the
instant invention may be obtained from a wide variety of commercial
vendors, e.g., EMD Millipore (Billerica, Mass.); Becton Dickinson
(Franklin Lakes, N.J.); Life Technologies (Carlsbad, Calif.);
Thermo Fisher Scientific (Pittsburgh, Pa.); and Sigma Aldrich (St.
Louis, Mo.)
[0091] In addition to using and augmenting or otherwise modifying
commercially available, "ready-to-use" liquid or solid culture
media for use in the methods of the instant invention, it is also
contemplated herein that bacterial culture media may be custom
designed using raw materials by one of skill in the art. For
example, culture media may be custom formulated for use in the
methods of the instant invention starting with a wide variety of
commercially available raw materials, base materials, and culture
media supplements. Such raw materials are familiar to one of skill
in the art and include, e.g., peptones, yeast extracts, as well as
defined amino acids, carbon sources, and vitamin components.
Similarly, supplements employed for culturing particular bacterial
pathogens are familiar to one of skill in the art and include,
e.g., defined carbon sources, essential amino acids, and essential
micronutrients and vitamins.
[0092] It is contemplated herein that one or more culture features
may be modified to more closely approximate various physiological
aspects associated with a bacterial infection in vivo. However,
regardless of how well the media recapitulates in vivo conditions,
it is understood herein that varying the bacterial physiological
gene expression in phage harvesting and/or isolation cultures
according to the methods of the invention will enhance and
diversify total phage isolation. Notably, the results provided in
the examples provided herein below do not include gene expression
data, however, one of skill in the art will appreciate that the
varied results, at least in part, likely reflect changes in gene
expression. Additionally, the results presented here, which do
demonstrate conditionally dependent phage isolation and
conditionally dependent phage host-infection, were obtained using
numerous bacterial strains and several different bacterial species.
The precise mechanism in each species, and possibly in each strain,
is likely unique, yet the overall method is robust enough for broad
application across numerous strains and species such that the exact
mechanism(s) is a nuance.
[0093] As discussed in detail below, one or more culture
modifications that may be made include but are not limited to:
varying the temperature of the cultures (e.g., more or less than
37.degree. C.), modifying (e.g., reducing) the level of iron in the
cultures; adding whole or fractionated serum to the cultures;
adding whole or fractionated plasma to the cultures; adding organ
homogenates such as mouse or other mammalian liver, heart, spleen,
and kidney homogenates; modifying the concentration and identity of
nutrients in the culture (e.g., nutrient starvation and/or the
inclusion of exclusively phosphorylated sugars); modifying the pH
of the cultures to stress the bacteria or buffering the culture
conditions with a buffer such as carbonic-acid-bicarbonate;
modifying the level of magnesium in the cultures (e.g., magnesium
limitation); modifying the level of carbon dioxide and/or oxygen
(e.g., culturing in anaerobic or microaerophilic conditions) and/or
modifying (e.g., increasing) the levels of known inducers of
bacterial virulence factors such as activated charcoal,
phosphorylated sugars, and/or cholesterol. Gene expression may also
be altered to more closely mimic infection pattern by modifying the
culture conditions to promote biofilm formation. See e.g., Boyce,
J. D., Cullen, P. A., & Adler, B. (2004). Genomic-scale
Analysis of Bacterial Gene and Protein Expression in the Host.
Emerging Infectious Diseases, 10(8), 1357-1362.
Iron Limitation
[0094] It has been reported that bacterial gene expression may be
modified by reducing iron levels in bacterial cultures. See
Paustain et al, Infect. Immun 2001 69:4109-4115. Thus, it is
contemplated herein that the conditional cultures of the instant
invention may be modified to include iron levels in only trace
amounts, e.g., well below the range of iron for bacterial growth of
0.3 .mu.M to 1.8 .mu.M. Free iron in the host is nearly
non-existent. Iron limiting conditions, and/or iron supplied only
in the form a heme, mimics the free-iron concentration and source
of iron present during a bona-fide host infection in a human host.
For example, in a particular embodiment, one of skill in the art
will appreciate that "iron free media" may be created using a
defined rich media that eliminates all but trace iron supplied by
the trace amounts in the individual components of the defined rich
media. Chelators can be added to further reduce the free iron,
and/or iron may then be added back in the form of heme to mirror
the iron levels and sources of iron present in a mammalian
host.
Inducers of Bacterial Virulence Factors
[0095] In various embodiments, the methods of the instant invention
may comprise mixing raw environmental samples with microbial
culture media that is augmented with additives capable of inducing
bacterial virulence factors. These factors may or may not also have
nutritive value for the bacteria. As understood herein, "bacterial
virulence factors" are those gene products of a bacterial pathogen
which enable it to invade a host, colonize a host, survive within a
host, and/or cause disease within a host. For example, these
factors include proteins produced by the bacteria which can
facilitate bacterial adhesion to host cells, colonization of the
host, invasion of host cells, and/or toxins that directly harm the
host. They include fats, carbohydrates, proteins and toxins, found
on the surface of the bacteria, in the bacterial cell wall or
membrane, or secreted by the bacteria. See, e.g., Wu et al, Current
Opinion in Chemical Biology 2008, 12:93-101. Surface features such
as these can also serve as phage receptors, and many of these
features are not expressed outside of a host or are massively
upregulated during the infection of a host. Thus, these factors are
often not functionally present in bacteria growing in vitro, and
inducing their expression during phage harvesting and/or isolation
allows for finding phages that may use them as receptors and
therefore may possess enhanced therapeutic efficacy. These
additives include but are not limited to: Fetal Bovine Serum (FBS),
Glucose-6-Phosphate, activated charcoal, and cholesterol.
Modification of culture levels of magnesium have also been reported
as affecting the expression of certain virulence factors. See Guina
et al, J. Am Soc Mass Spectrom. 2003 14:742-751, the entire
contents of which are incorporated by reference herein.
[0096] Alterations in bacterial gene expression during the
infection of a host may also include the down-regulation of certain
genes expressing bacterial surface features. Similarly, the
augmented cultures outlined here, intended to produce more
host-like physiological states in the bacterial pathogens, may also
yield bacteria that specifically "turn off" or downregulate the
expression of surface features that are present during traditional
in vitro culture growth, but are not present or are downregulated
during infection of a host. These now deactivated surface features,
when present, may skew or cause the selection of, or favor the
isolation of, phages that preferentially infect in vitro adapted
bacteria.
[0097] Thus, these in vitro associated surface features may serve a
decoy function, and their simple absence might enhance the
isolation of phages against host-adapted bacteria. This strategy of
attempting to turn off receptors and find fewer phages against in
vitro adapted bacteria runs completely counter to current methods
looking to maximize the identification of so called "broad host
range" phages in classical broth cultures in vitro. Thus, it is
contemplated herein that, using the methods of the instant
invention, one would find less of such bacteria in some conditional
cultures across the set of the conditional cultures, with the
express purpose of finding phages better-suited to infecting
host-adapted bacteria. Specifically, the down-regulation of decoy
or specifically in vitro surface features may enhance the isolation
of phages that use surface receptors present during both in vitro
growth and during an infection, because removing the decoy
receptors and therefore the competition of phages that use these
receptors normally downregulated during the infection of a host,
will enhance the isolation of phages better-suited to infecting
host-adapted bacteria. These augmented cultures are then grown at
variable temperatures, and under aerobic and anaerobic conditions
for the required period of time, e.g. over-night, depending on the
strain and condition needs. The phage-rich supernatant of these
cultures will be specifically enriched for phages that infect
host-adapted bacterial pathogens. These supernatants are then
subjected to plaquing assays to identify phages against the target
pathogen, if present. For example, methods for analyzing the
supernatants include, but are not limited to, traditional
plate-based plaquing assays, liquid assays, or high throughput
assays. These classical plaquing assays are performed under the
same conditions as the augmented cultures so as to maintain the
bacterial pathogen in the same physiological state as during the
initial isolation. Phages harvested under these conditions can then
be screened for therapeutic or diagnostic efficacy.
Serum and Plasma
[0098] It is contemplated herein that adding whole or fractionated
serum or plasma to bacterial cultures may induce gene expression
changes in bacterial pathogens, including changes in virulence
factor expression. These changes in gene expression and/or
virulence factor expression may include surface proteins or other
surface features, and therefore phage receptors. In various
embodiments, whole or fractionated serum or plasma for use in the
methods of the instant invention are selected from the group
consisting of those derived from humans and animals. Serum may be
obtained from a variety of commercial vendors, including major
distributors such as Fisher and Sigma Aldrich.
Nutrients
[0099] Infection of a host presents a bacterial pathogen with in
vivo conditions which are nutrient-deficient. Reports indicate that
nutrient limited conditions can trigger expression of various
genes, including several encoding outer membrane proteins, which
may serve as virulence factors, and may contribute to the
regulation of colonization factors. See e.g., Paustian et al., J.
Bacteriol. 2002, Vol. 184:3734-3739. It is contemplated herein that
these outer membrane proteins may also serve as phage receptors.
Thus, in a particular embodiment, the methods of the present
invention contemplate culturing environmental samples comprising
bacteriophage and a bacterial pathogen in minimal medium. The term
"minimal medium" is familiar to one of skill in the art and
includes but is not limited to limiting the carbon source,
available proteins and/or amino acids, fatty acids, vitamins, and
crude extracts (e.g. yeast extracts, brain extracts, etc.)
Different types of minimal media may be obtained from commercial
vendors and include, e.g., M9 minimal media (Thermo Fisher
Scientific) and custom derivatives thereof.
[0100] Additionally, nutrients may be added that mimic the
available nutrients in a host environment, such as using
phosphorylated sugars, triglycerides, or DNA as carbon sources, and
adding iron in the form of heme are examples of supplying nutrients
as they exist in the host-environment. A variety of different cell
culture media (commercially available or custom formulated) may be
used.
[0101] Regardless of how effective conditional cultures are in
generating host-adapted bacteria, using conditional cultures
according to the instant invention may result in bacterial gene
expression changes and/or produce direct actions on the phage
particles themselves that will alter phage competition dynamics
across the conditions, thus producing differentially enriched
populations of phages from the original environmental sample across
the conditional cultures, allowing for enhanced or even maximal
phage harvesting from the original environmental sample.
pH
[0102] Possible virulence factors for bacterial pathogens, e.g.,
enteric pathogens, may be induced by an acidic environment, e.g.,
Heliobacter pylori virulence factors may be influenced by the pH of
the culture medium. See Merrell et al., Infect. Immun. 2003 Vol.
71: 3529-35379; Karita M. et al. Infect. Immun. 1996 64:4501-4507;
Ang et al., Infect. Immun. 2001 69:1679-1686. Thus, it is
contemplated herein that modifying the pH of culture conditions may
be used to mimic an in vivo infection. In a particular embodiment,
the pH of the medium may be titrated below about 7.2, e.g., to
about pH 5.5 or below, and/or within a range of 5.5-8.5. In
addition to subjecting the pathogens to acid stress, a major
endogenous buffer in the mammalian host is the
carbonic-acid-carbonate buffering system in blood. Using this
buffering system in vitro may better mimic the host
environment.
Incubation Conditions
[0103] In various embodiments, conventional in vitro bacterial
culture conditions may be modified to more closely resemble
conditions that bacteria will encounter during an in vivo infection
of a host. These conditions may be mimicked by modifying not only
the formulation of the culture media, but also by modifying the
cell culture incubator conditions, e.g., the temperature, humidity,
osmotic pressure, carbon dioxide, and oxygen content of the
incubator atmosphere as discussed in detail below.
Temperature
[0104] Conventionally, bacterial cultures are typically maintained
at temperatures which provide optimum growth conditions. Typically,
for pathogenic bacteria this is 37.degree. C. (98.6 F). While the
conditional cultures of the instant invention may be modified in
other ways and still cultured at 37.degree. C., it is also
contemplated herein, however, that different repertoires of
bacterial gene expression may be induced by manipulating the
culture temperature above or below 37.degree. C. (e.g. up to about
the maximum growth temperature or down to about the minimum growth
temperature). This genetic response to temperature change may be
useful to enhance the expression of one or more virulence factors
in a bacterial pathogen, or may result in other surface feature
changes thereby altering potential phage receptors. Specifically,
heat-stress is used by the host during infection and the
development of fever. Growing the bacterial cultures at 42.degree.
C. alone or in combination with other culture changes, may produce
physiological conditions that better mirror those of a bona-fide
infection. Phages isolated on these bacteria may have better
therapeutic potential. Data provided in the examples include
experiments performed at 37.degree. C. as well as 25.degree. C.
(room temperature).
Carbon Dioxide and Oxygen
[0105] One of skill in the art will appreciate that bacteria are
typically classified as aerobes, microaerophiles, obligate
anaerobes, aerotolerant, and facultative organisms. To this end, it
is contemplated herein that the methods of the present invention
may comprise modifying the level of oxygen and/or carbon dioxide in
bacterial cultures to more closely mimic in vivo infections of
obligate anaerobes or microaerophiles. Indeed, it has been reported
that microaerophilic conditions are associated with the activation
of H. pylori virulence genes. See Cottet et al., J. Biol. Chem.,
2002 277:33978-339986. Additionally, bacterial pathogens growing in
localized wounds, in organs (including the liver and spleen), or
systemically in blood are actually in microaerophilic environments.
Thus, mimicking this condition will better mirror the oxygen
tension present in an actual infection. Thus, it is contemplated
herein that in various embodiments, the conditional cultures of the
instant invention may be grown in incubators from about 0-20%
O.sub.2. and/or from about 0-7% CO.sub.2, particularly at less than
2% O.sub.2 and/or at 5% CO.sub.2.
Biofilm and Planktonic Growth
[0106] One of skill in the art will appreciate that conventional
phage harvesting methods rely on the use of planktonic cells to
isolate and harvest phages. Literature reports indicate, however,
that gene expression patterns are different in biofilms and in
planktonic bacteria. See Tremoulet et al., FEMS Microbiol Lett 2002
210:25-31. Thus, it is contemplated herein that gene expression
patterns that mirror infection could occur not only in cultures of
planktonic cells that have host-like surface features, but also in
bacteria growing in biofilms. Thus, both types of cultures are
encompassed according to the methods of the instant invention.
[0107] Indeed, bacteria growing in numerous types of infections,
including wound infections, surgical hardware associated
infections, and even some organ infections, are known to grow as a
biofilm. Additionally, the biofilm may also confer antibiotic
resistance, exacerbating the infection. Thus, in a particular
embodiment, it is contemplated herein that the methods of the
instant invention comprise using bacteria first grown in a biofilm
(e.g., on biotic and/or abiotic surfaces), to drive gene expression
and surface protein expression to mirror the bacterial surface
features present in a biofilm growing during an actual infection,
and then using that biofilm for phage isolation in the methods of
the invention.
[0108] In a particular embodiment, the culture condition induces
the formation of bacterial biofilm. These culture modifications
include but are not limited to increasing culture incubation time
up to several days, e.g., from about 18 hours to about 7 days
(contrary to typical culture growth of 18 hours or less), not
agitating the culture, and modifying the media to one that supports
biofilm production, e.g., pro-biofilm media often contains stronger
buffers to detoxify acidic waste products and the increased
concentration of carbon sources. In another embodiment, biofilm
formation may be induced by culturing the bacteria on biotic
surfaces and/or abiotic surfaces such as collagen coated surfaces,
bone, and surfaces coated with host cells lysates, as well as
stainless steel, titanium, plastic, and aluminum. In a particular
embodiment, biotic surfaces may comprise complex host extracts. As
understood herein, "complex host extracts" may comprise, e.g.,
crude, unpurified homogenates/lysates of tissue culture cells,
e.g., mouse and human cells, and/or homogenized muscle/bone from
mice and rats.
[0109] Biofilm matrix material and bacterial surface features
within the biofilm can change as the biofilm is grown on different
surfaces. Thus, in a particular embodiment, it is contemplated
herein that the methods of the instant invention may comprise
culturing bacteria on different biotic and abiotic surfaces in
order to more closely reflect conditions associated with both body
surface biofilm infections, as well as surgical hardware associated
infections in vivo.
Measuring Gene Expression
[0110] In a particular embodiment, it is contemplated herein that
modifying the bacterial culture conditions according to the methods
of the instant invention may trigger an expression pattern of one
or more genes in said infectious pathogenic bacteria in vitro that
are similar to an expression pattern of said one or more genes in
said infectious pathogenic bacteria during infection of said host
in vivo. Specifically, it is contemplated herein that by modulating
culture conditions to more closely resemble the physiological and
biochemical environment in vivo, gene expression in cultured
bacteria cells may be manipulated to more closely reflect the
bacterial proteome in vivo, including the surface features that may
serve as phage receptors in vivo.
[0111] As used herein, the phrase, "trigger an expression pattern
of one or more genes" and like terms refers to the ability of one
or more culture conditions to drive the expression of one or more
genes in the cultured bacteria. This includes the ability of the
culture condition(s) to upregulate or downregulate gene expression
levels. For example, as discussed above, it is contemplated herein
that the methods of the instant invention may promote or upregulate
the expression of genes encoding bacterial surface features found
in host-adapted bacteria, but not expressed in conventional
cultures. It is also contemplated herein that the methods of the
instant invention may turn off or reduce the expression of one or
more bacterial surface proteins that may act as phage receptors in
vitro, but actually do not play a major role in the interaction
between phage and bacteria in vivo as in vivo these receptors are
also downregulated and/or are not present. It is contemplated
herein that the down-regulation of such "decoy" receptors in the
conditional cultures of the instant invention may facilitate the
identification of clinically more useful phages, e.g., by
permitting binding to less abundant and/or obscure bacterial
surface receptors that are present during both in vitro and in vivo
growth.
[0112] Notably, the fact that a phage may not infect a bacterial
strain grown in vitro, or may not infect in a manner that would
predict clinical utility, but can nevertheless provide a clinical
benefit against the same bacterial strain grown in vivo, or under
host-like conditions, is at odds with current and conventional
methods for identifying phages for therapeutic use. In addition,
according to the tenets of conventional methods of phage
harvesting, obscure bacterial surface receptors might be deemed of
little interest when phages are identified in vitro. Indeed, it is
against all current strategies used in vitro for phage harvesting
to purposefully get rid of receptors on the surface of bacteria
that allow for robust phage predation in vitro. In contrast, in a
particular embodiment, the methods of the instant invention are
designed to induce the expression of bacterial surface receptors
which do not allow for phage predation in vitro.
[0113] It is also contemplated herein that, in a particular
embodiment, harvesting wild-phages from conditional cultures using
the methods of the present invention may permit the isolation of
phages that use atypical receptors and/or which will have different
host ranges relative to phages isolated from traditional (e.g.,
37.degree. C.) cultures. While of scientific interest, and might be
analyzed using conventional methods, it is not necessary to
characterize the changes on the bacterial surface which occur when
the bacteria are cultured according the methods of the instant
invention, as the objectives of the instant invention include
improved methods of isolating phages from environmental sources,
including phages which may be more likely to have a therapeutic use
against infectious pathogenic bacteria in vivo. Indeed, it is
understood herein that phages identified according to the methods
of the instant invention may be used to provide enhanced
therapeutic efficacy without necessarily first understanding the
mechanism of action behind the increased therapeutic efficacy.
[0114] In fact, one of skill in the art will appreciate that the
high-throughput and empirically determined results provided
according to the methods of the instant invention run completely
counter to current methods. Nevertheless, so long as the phages
isolated according to the methods of the instant invention are
characterized as safe for human use, they can be used to build
robust and diverse phage libraries directed against infectious
pathogenic bacteria, including but not limited to MDR ESKAPE
bacterial pathogens selected from the group consisting of
Enterococcus faecium, Staphylococcus aureus, Kiebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter
spp. It is contemplated herein that these phage libraries may be
created and the phages used for various industrial and/or
therapeutic purposes, e.g., in library-to-cocktail and/or other
therapeutic phage modalities according to conventional methods.
See, e.g., U.S. Ser. No. 15/628,368, the entire contents of which
are incorporated by reference herein.
Methods of Measuring Effects of Phage on Bacteria
[0115] Methods for identifying the ability of a phage to "infect
and kill" target bacteria in the conditional cultures of the
instant invention may be performed using conventional methods. For
example, the assay may comprise determining the effectiveness of
one or more phages to prevent bacterial growth in the conditional
culture media (which otherwise supports robust bacterial growth.)
This may include, for example, performing conventional phage plaque
assays or spot assays where the effectiveness of phage or various
phage combinations to prevent bacterial growth can be evaluated in
conditional cultures on solid agar or semi-solid medium media. Such
techniques are familiar to one of skill in the art. See, e.g.,
Sambrook, J., E. F. Fritsch and T. Maniatis (1989). "Molecular
Cloning: A Laboratory Manual. 2nd ed." Cold Spring Harbor Press,
Cold Spring Harbor, N.Y.
[0116] Other methods for identifying the ability of a phage to
"infect and kill" target bacteria may comprise the use of "phage
efficacy assays" which comprise growing cultures of a targeted
bacterial pathogen with individual phages or phage combinations and
analyzing bactericidal activity against the targeted bacterial
pathogen, wherein a suitable delay in bacterial growth and/or the
lack of appearance of phage-resistant bacterial growth in the
culture indicates that the phage(s) may be therapeutically
effective against the targeted bacterial pathogen in vivo.
[0117] High-throughput methodologies comprising the use of
microtiter plates and liquid media for running multiple
simultaneous assays and cultures are also contemplated herein. For
example, a clinical isolate of a bacterial pathogen may be raised
in conditional cultures or as biofilms as described herein, and
then a culture inoculum may be aliquoted to phage dilutions in
wells of a 96 well-plate, or the biofilm is first grown in the
plate. The interaction between the bacteria and phage is then
monitored for evidence of bacterial growth delay, and or
destruction of the biofilm. In a particular embodiment, the delay
in bacterial growth and/or the lack of appearance of
phage-resistant bacterial growth, or the destruction of the
biofilm, may be monitored comprising the use of a high-throughput
photometric assays and liquid media, and/or microscopic analysis of
biofilm destruction. In particular embodiments, the photometric
assay may be, e.g., fluorescence, absorption, or transmission
assays. In a particular embodiment, the photometric assay comprises
a step wherein an additive such as tetrazolium dye is used to cause
and/or enhance the photometric signal detection.
[0118] For example, in a particular embodiment, such assays include
phage efficacy assays monitoring the delay in bacterial growth,
called a growth "hold-time," which can be used to determine the
lytic activity of individual phages or compounded phage cocktails
using an automated, high throughput, indirect liquid lysis assay to
evaluate phage bactericidal activity using an OMNILOG system (Henry
M, et al. 2012. Bacteriophage 2:159-167). Such assay is described
in detail in U.S. Ser. No. 15/628,368, the entire contents of which
are incorporated by reference herein. Biofilm destruction assays
may be monitored by direct microscopy or indirect measurements
using crystal violet staining followed by photometric analysis
according to conventional methods.
[0119] As used herein, the terms "desirable delay in bacterial
growth", "suitable delay in bacterial growth", "growth hold time",
"lack of appearance of phage-resistant bacterial growth", "biofilm
destruction" and like terms is understood to relate to the
effectiveness of a phage, phage combination, or phage cocktail to
prevent bacterial growth for a given amount of time in culture or
to degrade biofilm structures in culture. In a particular
embodiment, this includes bacterial growth in the liquid culture
environment as described in U.S. Ser. No. 15/628,368. Typically, in
this assay, growth hold-time indicative of a promising phage is
from about 4 to about 8 hours. In a particular embodiment, the
growth hold time of a promising phage cocktail, assembled from
individual phages deemed as promising, may be a minimum hold-time
of about 12 hours to about 18 hours or longer without limit. In
other particular embodiments, the growth hold time of a phage or
phage cocktail may be from about 15, 16, 17, 18, 19, or 20 hours.
In another embodiment, cocktail hold-times of less than 12 hours
may have therapeutic efficacy.
[0120] In yet another embodiment, the growth hold-time of a
promising individual phage may be zero or undetectable and only
when this type of phage is included with another phage deemed to be
promising, or as a new constituent of a cocktail deemed to be
promising, will the activity and necessity of such a phage become
detectable. In such situations, phages of this type, which have
undetectable or nearly undetectable activities on their own, can
surprisingly add to a synergistic hold-time when included in
promising phage cocktails.
[0121] It is contemplated herein that the methods of the instant
invention can be used to identify a phage or a phage combination,
including synergistic phage combinations, which can produce a
complete or nearly complete growth arrest of the bacterial
pathogen. This may be evident from a growth hold time from about
16-48 hours or more.
[0122] One of skill in the art will appreciate that promising
growth hold times, including minimum hold times, and growth hold
times indicative of complete or nearly complete growth arrest, may
vary depending on the species of bacteria, e.g., some bacterial
species typically grow more slowly than other bacteria. Promising
growth hold times of bacteria under investigation may be easily
discerned according to the methods of the instant invention without
undue experimentation.
[0123] In addition to hold time in liquid cultures, biofilm
destruction may also be monitored and individual phages, phage
cocktails, synergistic phage cocktails, and promising phages with
no detectable activity on their own, may be scrutinized as before
as with liquid cultures, but the read out for potential therapeutic
efficacy here is the ability of individual phages or phage
cocktails to degrade or destroy biofilms. Biofilm destruction is
scored by direct monitoring using microscopy or indirect
measurements using staining techniques according to conventional
methods.
[0124] As understood herein, a "subject", "subject in need thereof"
and like terms encompass any organism, e.g., any animal or human,
that may be suffering from a bacterial infection, particularly an
infection caused by a MDR bacteria.
[0125] As used herein, a "clinical isolate" is a pathogenic
bacteria harvested from human or animals during course of
pathogenesis or gradual progression of a specific disease, e.g., an
infectious bacterial pathogen that was isolated from a bona-fide
human infection.
[0126] As understood herein, a "bona-fide human infection" refers
to a bacterial infection, which produces pathogenesis in humans,
including, e.g., a symptomatic infection that requires medical
intervention, including culturing the infectious bacterial
strain.
[0127] As used herein, the term "infectious pathogenic bacteria"
refers to a bacterial strain capable of causing disease or a
detectable pathology within a human or animal. In a particular
embodiment, the methods of the instant invention may be employed
with any bacterial pathogen, including but not limited to pathogens
that display differential gene expression in a host vs in vitro
growth. One of skill in the art will appreciate that all
facultative pathogens do this, including but not limited to, all of
the ESKAPE pathogens. Thus, bacteria that may be treated include,
but are not limited to the "ESKAPE" pathogens (Enterococcus
faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter
baumannii, Pseudomonas aeruginosa, and Enterobacter sp), which are
often nosocomial in nature and can cause severe local and systemic
infections. Specifically, these include, e.g.,
methicillin-resistant Staphylococcus aureus (MRSA);
vancomycin-resistant Enterococcus faecium (VRE);
carbapenem-resistant Klebsiella pneumonia (NDM-1); MDR-Pseudomonas
aeruginosa; and MDR-Acinetobacter baumannih.
[0128] Among the ESKAPE pathogens, A. baumannii is a Gram-negative,
encapsulated, opportunistic pathogen that is easily spread in
hospital intensive care units. For example, A. baumannii infections
are typically found in the respiratory tract, urinary tract, and
wounds. Many A. baumannii clinical isolates are also MDR, which
severely restricts the available treatment options, with
untreatable infections in traumatic wounds often resulting in
prolonged healing times, the need for extensive surgical
debridement, and in some cases the further or complete amputation
of limbs. Notably, blast-related injuries in military populations
are associated with significant tissue destruction with concomitant
extensive blood loss and therefore these injuries are at high risk
for infectious complications. One of skill in the art will
appreciate that given the ability for A. baumannii and other MDR
ESKAPE pathogens to colonize and survive in a host of environmental
settings, there is an urgent need for new therapeutics against
these pathogens.
[0129] Any type of bacterial contamination may be treated using the
methods, phage libraries, and compositions of the instant
invention. Particularly, bacterial infections to be treated using
the compositions, libraries, and methods of the instant invention
may include any infection by a bacterial pathogen that poses a
health threat to a subject. In a particular embodiment, bacteria
for treatment according to the methods of the present invention
include, but are not limited to, multidrug resistant bacterial
strains. As understood herein, the terms, "multidrug resistant",
"multi drug resistant", "multi drug resistance", "MDR" and like
terms may be used interchangeably herein, and are familiar to one
of skill in the art, i.e., a multidrug resistant bacteria is an
organism that demonstrates resistance to multiple different
antibacterial drugs, e.g., antibiotics; and more specifically,
resistance to multiple different classes of antibiotics. It is
understood herein that bacterial infections to be treated comprise
bacteria in biofilm and/or planktonic growth modes.
[0130] One of skill in the art will appreciate that bacterial
infections to be treated using the compositions, libraries, and
methods of the instant invention include any type of bacterial
infection in a subject. These include, for example, not only
infections that may be associated with wounds, but also non-wounds,
e.g., infections that might arise without underlying trauma or any
other type of bodily injury, traumatic or otherwise. These
infections may include local infections, e.g. a respiratory
infection or an internal or external abscess that progresses to a
systemic infection. Infections that may be treated according to the
methods of the instant invention also include infected surgical
wounds, e.g., "post-surgical" infections that may arise in a
subject after and/or resulting from a surgical procedure or any
other kind of medical or surgical treatment or intervention, e.g.,
a catheterization procedure, or surgical implantation of a medical
device, prosthetic, or other foreign object into a subject,
etc.
[0131] One of skill in the art will appreciate the myriad other
therapeutic uses for the compositions of the instant invention
given that the compositions can be administered both topically and
systemically, e.g. via IV or IM injections, or injected into the
peritoneal cavity. They may be provided as aerosols, or in any
other manner that is pharmaceutically suitable. The types of
infections that can be treated also include, for example,
infections associated with burns, ulcers, systemic bacteremia,
septicemia, inflammatory urologic disease, infections associated
with cystic fibrosis, abscesses, empyema, suppurative lung
diseases, as well as infections in other internal organs, including
but not limited to infections in the liver, spleen, kidney,
bladder, lungs etc.
[0132] As used herein, the term "sample comprising phages", "a
sample of phages" and like terms refer to a sample comprising
phages obtained from any source. It is understood by those of skill
in the art that phages are omnipresent; one can expect phages to be
present in any sample taken from a place where bacteria exist. This
includes but is not limited to samples obtained from nature as well
as man-made sources. The sample may comprise any number and
combination of "wild" phages (phages found in nature), previously
unidentified phages, or phages that have been previously isolated.
Samples comprising phages obtained from academia and commercial or
noncommercial sources are included in this definition. Thus, as
understood herein, a "sample comprising phages" for assay according
to the methods of the instant invention may be obtained by
harvesting phages from a variety of diverse environmental sources.
Such samples may comprise more than one phage, i.e., the sample may
comprise mixed phages. Samples of phages may be taken from a wide
variety of different places where phage may be found in the
environment, including, but not limited to, any place where
bacteria are likely to thrive. In fact, phages are universally
abundant in the environment. Samples of phages include but are not
limited to samples acquired from diverse environmental sources,
including samples which comprise uncharacterized "wild phages", as
well as samples of characterized phages that may be obtained from
laboratories or commercial vendors.
[0133] One of skill in the art will appreciate the myriad sources
of phage that may be assayed according to the methods of the
instant invention. For example, possible sources include, but are
not limited to, natural sources in the environment such as soil and
water, as well as man-made sources such as untreated sewage water
and water from waste-water treatment plants. Clinical samples from
infected subjects (e.g., human patients, animals or any other
species) may also serve as a source of phage. In a particular
embodiment, diverse environmental sources of phage may be selected
from the group consisting of soil, water treatment plants, raw
sewage, sea water, lakes, rivers, streams, standing cesspools,
animal intestines, human intestines, manure or other fecal matter,
organic substrates, biofilms, and medical/hospital sources. Phage
may be sourced anywhere from a variety of diverse locations around
the globe, e.g., within the US and internationally.
Bacteriophage and Bacterial Libraries
[0134] As discussed above, due to the upregulation and
downregulation of different proteins within bacteria grown in
host-like conditions, it is contemplated herein that by harvesting
wild phages from conditional cultures, it will be possible to
isolate phages from the environment that use atypical receptors and
will have different host ranges relative to phages isolated from
traditional 37.degree. C. cultures. Ideally, the methods of
conditional phage isolation as contemplated herein will select for
phages that are better suited to infecting bacteria growing within
a mammalian, e.g., human host, and thus permit the creation of
therapeutic phage cocktails which can provide enhanced therapeutic
efficacy.
[0135] In view of the foregoing, the identification of phages
according to the methods of the instant invention also permits the
creation of phage libraries of enhanced richness. In particular
embodiments, the methods described herein permit the creation of
enhanced phage libraries against bacterial pathogens of interest
comprising individual phages that have distinct but overlapping
host ranges. Such a library allows for maximal coverage of
clinically relevant bacterial strains, including MDR bacterial
pathogens.
[0136] Thus, in another aspect, the invention relates to
bacteriophage libraries comprising the bacteriophage identified
according to the methods of the instant invention. Specifically, it
is contemplated herein that the host range and plaque morphology of
phages grown up in conditional cultures may be compared to that of
phages harvested under classical conditions and used to compile a
robust library of diverse bacteriophage against different bacterial
pathogens, including bacteriophage with enhanced propensity to
infect and kill infectious bacterial pathogens in vivo. In a
particular embodiment, the development of a large globally-sourced
library of phages (e.g., 200-500 phages/pathogen) against MDR
pathogens is contemplated herein. In a particular embodiment, the
library is against an MDR ESKAPE bacterial pathogen selected from
the group consisting of Enterococcus faecium, Staphylococcus
aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa, and Enterobacter spp. The creation of such libraries
requires diverse collections of MDR ESKAPE pathogens and, ideally,
access to extremely contaminated water sources. In this regard,
large collections of diverse MDR ESKAPE pathogens have already been
obtained and are available for use according to the methods of the
instant invention as hosts for phage isolation; contaminated
environmental water sources may be collected from a diversity of
sources. The host ranges of the phages in these libraries will be
characterized and phages with broad host ranges will also be
sequenced.
[0137] One of skill in the art will appreciate that phages exist as
a population centered-around a consensus genome sequence, but there
is an n-dimensional distribution around that consensus sequence.
Thus, in a particular embodiment, it is contemplated herein that
starting with a diverse phage library, particularly in high
concentration, will create an enormous level of phage sequence
diversity to bring to bear against the problem of MDR resistance.
As discussed above, it is contemplated herein that the phage
libraries of the instant invention may be sourced from around the
globe. This is particularly relevant with regard to the design of
phage therapeutics to treat military service members who routinely
encounter bacterial pathogens from global sources. Indeed, phages
to those global pathogens are best found in the environment
adjacent to the site of the acquired infection. Thus, it is
contemplated herein that by harvesting phages from a variety of
sources from around the globe, and assaying these phages using
conditional cultures according to the methods of the instant
invention, large and genetically diverse bacteriophage libraries
may be successfully generated for use in creating phage
therapeutics.
[0138] The global and conditional phage libraries created here can
also serve as powerful starting materials for the development of
population level cocktails, engineered phages, and augment the
library-to-cocktail approach. Indeed, it is contemplated herein
that all current methodologies of developing therapeutic phage,
whether one is developing population level cocktails, engineered
phages, or employing a library-to-cocktail personalized therapeutic
approach, would benefit from methods designed to identify
additional phage that can infect and kill bacterial pathogens under
infection-associated physiological conditions, and thus provide the
means to create more robust phage libraries and therapeutics
against these pathogens. Thus, in particular embodiments, phage
libraries of the instant invention include but are not limited to
libraries of phage against MDR bacterial pathogens, including e.g.,
ESKAPE phage libraries. It is contemplated herein that these new,
more robust libraries can be created by using the methods of the
instant invention, and can serve as rich starting resources for the
continued development of other phage-based therapeutic
modalities.
[0139] As discussed herein, the methods of the instant invention
include creating unique and expanding sets of clinically relevant
bacterial pathogens, including but not limited to MDR bacterial
strains, and using these local MDR bacterial strains isolated from
the sources proximal to the phage harvesting efforts to increase
the chances of finding diverse and appropriate phages.
[0140] One of skill in the art will appreciate that, in addition to
the loss of receptor and receptor mutations that can block phage
infectivity, bacterial strains can become resistant to
phage-infection downstream of phage attachment and genome
injection. In addition, phage resistance is to be expected during
therapy. However, developing resistance to one phage may sensitize
the strain to another phage, and this resistance/sensitization is
extremely difficult to predict a priori. Phage libraries are tools
that enable the empirical selection of phages that work together
through rounds of resistance/sensitization.
[0141] Compositions and Methods of Treatment
[0142] It is contemplated herein that the creation of large
libraries of phages that are capable of infecting and killing
bacterial cultures which are phenotypically more consistent with
bacterial pathogens in vivo i.e., bacteria which in vivo display a
host-adapted gene expression pattern and surface-feature
repertoire, may effectively facilitate the enhanced development of
numerous phage therapeutic modalities, including natural phage
products, as well as phages engineered to carry antimicrobial cargo
or to possess expanded host-range. Thus, in another aspect, the
instant invention relates to compositions, including pharmaceutical
compositions, comprising one or more phages identified according to
the methods of the instant invention and which demonstrate enhanced
therapeutic efficacy. In a particular embodiment, the composition
is a "phage cocktail" comprising a plurality of phages against
infectious pathogenic bacteria.
[0143] The compositions of the instant invention, in a
pharmaceutically acceptable dosage form, may be administered to a
subject in a manner as deemed appropriate by an attending
physician. In particular embodiments, the compositions are
therapeutically effective compositions of very high titer and very
high purity, or of high titer and high purity, which are not found
in nature. Such degrees of purity and titer are familiar to one of
skill in the art, and are discussed at length in U.S. Ser. No.
15/628,368, the entire contents of which are incorporated by
reference herein.
[0144] Indeed, it is contemplated herein that the compositions
produced according to the methods of the instant invention may be
unique in composition as well as uniquely effective compared to
compositions made according to conventional methods. As used
herein, the term "composition" encompasses pharmaceutical
compositions comprising a plurality of purified phages, e.g., a
composition of the instant invention may be a "phage cocktail."
[0145] "Pharmaceutical compositions" are familiar to one of skill
in the art and typically comprise active pharmaceutical ingredients
formulated in combination with inactive ingredients selected from a
variety of conventional pharmaceutically acceptable excipients,
carriers, buffers, diluents, etc. Methods of formulating
pharmaceutical compositions are familiar to one of skill in the
art.
[0146] The term "pharmaceutically acceptable" is used to refer to a
non-toxic material that is compatible with a biological system such
as a cell, cell culture, tissue, or organism. Examples of
pharmaceutically acceptable excipients, carriers, buffers, diluents
etc. are familiar to one of skill in the art and can be found,
e.g., in Remington's Pharmaceutical Sciences (latest edition), Mack
Publishing Company, Easton, Pa. For example, pharmaceutically
acceptable excipients include, but are not limited to, wetting or
emulsifying agents, pH buffering substances, binders, stabilizers,
preservatives, bulking agents, adsorbents, disinfectants,
detergents, sugar alcohols, gelling or viscosity enhancing
additives, flavoring agents, and colors. Pharmaceutically
acceptable carriers include macromolecules such as proteins,
polysaccharides, polylactic acids, polyglycolic acids, polymeric
amino acids, amino acid copolymers, trehalose, lipid aggregates
(such as oil droplets or liposomes), and inactive virus particles.
Pharmaceutically acceptable diluents include, but are not limited
to, water, saline, and glycerol.
[0147] As understood by one of skill in the art, the type and
amount of pharmaceutically acceptable additional components
included in the pharmaceutical compositions of the instant
invention may vary, e.g., depending upon the desired route of
administration and desired physical state, solubility, stability,
and rate of in vivo release of the composition.
[0148] As contemplated herein, the pharmaceutical compositions of
the instant invention comprise an amount of phage in a unit of
weight or volume suitable for administration to a subject. In this
regard, it is noted herein that using conventional methods,
isolated phages are directed against in vitro adapted bacteria,
thus conventional methods typically employ very high titer of
phages for therapeutic use. In contrast, it is contemplated herein
that because phages identified using the methods of the instant
invention better match the in vivo physiology of the targeted
infections pathogenic bacteria, such phages may be able to be used
for therapeutic purposes in much lower titers.
[0149] The volume of the composition administered to a subject
(dosage unit) will depend on the method of administration and is
discernible by one of skill in the art. For example, in the case of
an injectable, the volume administered typically may be between 0.1
and 1.0 ml, e.g., approximately 0.5 ml. In another embodiment, up
to 10 ml may be delivered in conjunction with a saline IV.
[0150] For administration by intravenous, cutaneous, subcutaneous,
or other injection, a pharmaceutical formulation is typically in
the form of a pyrogen-free, parenterally acceptable aqueous
solution of suitable pH and stability, and may contain an isotonic
vehicle as well as pharmaceutical acceptable stabilizers,
preservatives, buffers, antioxidants, or other additives familiar
to one of skill in the art. It is understood herein that isotonic
properties of Ringer's solution make a suitable buffer for phage
cocktails, while "SM buffer" may be used for phage dilution and
storage. Of particular interest with phage therapeutics is the
removal or limitation of host bacterial components from the phage
cocktail preparation that may have deleterious effects on the host,
which include but are not limited to LPS, peptidoglycan, bacterial
toxins, and bacterial DNA. Therapeutic cocktail preparations can be
designed to contain these kinds of materials in amounts below
acceptable limits.
[0151] In another aspect, the present invention relates to methods
of treating a bacterial infection comprising administering to a
subject in need thereof an effective amount of a composition
comprising one or more phages identified according to the methods
of the instant invention. In a particular embodiment, the
composition is a pharmaceutical composition.
[0152] As understood herein, a "subject in need thereof" includes
any human or animal suffering from a bacterial infection, including
but not limited to a multidrug resistant bacterial infection.
[0153] As understood herein, terms such as "effective amount" and
"therapeutically effective amount" of a composition of the instant
invention, refer to an amount of a composition suitable to elicit a
therapeutically beneficial response in the subject, e.g., by
eradicating a bacterial pathogen in the subject and/or altering the
virulence or antibiotic susceptibility of surviving phage-resistant
bacterial pathogens and/or by providing an added benefit when a
composition of the instant invention is simultaneously administered
with either effective and/or ineffective antibiotics. Such response
may include e.g., preventing, ameliorating, treating, inhibiting,
and/or reducing one of more pathological conditions associated with
a bacterial infection. One of skill in the art will appreciate that
it is desirable that the initial dose of a phage cocktail of the
instant invention be sufficient to control the bacteria population
before it reaches a lethal threshold. Animal models suggest that
10.sup.9 to 10.sup.11 pfu/ml phage particles per dose would likely
be the maximum dosage tenable based on protein load presented
acutely to the liver in an adult (which would be scaled down in a
pediatric population, i.e., EU limited 10.sup.5 dosing discussed in
the below examples). It is suspected that this is a sufficient
acute bolus to reduce the bacterial burden sufficiently to
potentiate an immune response. Notably, phage "viremia" may be
measured in the blood after administration. Animal models suggest
that viremia is quite transient given the host immune response and
sequestration in the reticuloendothelial system (liver and
spleen).
[0154] Suitable effective amounts of the compositions of the
instant invention can be readily determined by one of skill in the
art and can depend upon the age, weight, species (if non-human) and
medical condition of the subject to be treated. In addition, one of
skill in the art will appreciate that the type of infection (e.g.,
systemic or localized), and the accessibility of the infection to
treatment may also impact the dosage amount that is deemed
effective. One of skill in the art will appreciate that initial
information may be gleaned in laboratory experiments and an
effective amount of a composition for humans subsequently
determined through dosing trials and routine experimentation.
[0155] It is contemplated herein that the compositions of the
instant invention may be administered to a subject by a variety of
routes according to conventional methods, including but not limited
to systemic, parenteral (e.g., by intracisternal injection and
infusion techniques), intradermal, transmembranal, transdermal
(including topical), intravesicular, intramuscular,
intraperitoneal, intravenous, intra-arterial, intralesional,
subcutaneous, oral, and intranasal (e.g., inhalation of an
aerosolized composition) routes of administration. Administration
can also be by continuous infusion or bolus injection.
[0156] In addition, the compositions of the instant invention can
be administered in a variety of dosage forms. These include, e.g.,
liquid preparations and suspensions, including preparations for
parenteral, subcutaneous, intradermal, intramuscular,
intraperitoneal or intravenous administration (e.g., injectable
administration), such as sterile isotonic aqueous solutions,
suspensions, emulsions or viscous compositions that may be buffered
to a selected pH. In a particular embodiment, it is contemplated
herein that the compositions of the instant invention are
administered to a subject as an injectable, including but not
limited to injectable compositions for delivery by intramuscular,
intravenous, subcutaneous, or transdermal injection. Administration
by inhalation of an aerosolized composition is also contemplated
herein. Such compositions may be formulated using a variety of
pharmaceutical excipients, carriers or diluents familiar to one of
skill in the art.
[0157] In another particular embodiment, the compositions of the
instant invention, and/or pharmaceutical formulations administered
in conjunction therewith, e.g., antibiotics, may be administered
orally. Oral formulations for administration according to the
methods of the present invention may include a variety of dosage
forms, e.g., solutions, powders, suspensions, tablets, pills,
capsules, caplets, sustained release formulations, or preparations
which are time-released or which have a liquid filling, e.g.,
gelatin covered liquid, whereby the gelatin is dissolved in the
stomach for delivery to the gut. Such formulations may include a
variety of pharmaceutically acceptable excipients described herein,
including but not limited to mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, and magnesium
carbonate.
[0158] In a particular embodiment, it is contemplated herein that a
composition for oral administration may be a liquid formulation.
Such formulations may comprise a pharmaceutically acceptable
thickening agent which can create a composition with enhanced
viscosity which facilitates mucosal delivery of the active agent,
e.g., by providing extended contact with the lining of the stomach.
Such viscous compositions may be made by one of skill in the art
employing conventional methods and employing pharmaceutical
excipients and reagents, e.g., methylcellulose, xanthan gum,
carboxymethyl cellulose, hydroxypropyl cellulose, and carbomer.
[0159] Other dosage forms suitable for nasal or respiratory
(mucosal) administration, e.g., in the form of a squeeze spray
dispenser, pump dispenser, nebulizer, or aerosol dispenser, are
contemplated herein. Dosage forms suitable for rectal or vaginal
delivery are also contemplated herein. Where appropriate,
compositions for use with the methods of the instant invention may
also be lyophilized and may be delivered to a subject with or
without rehydration using conventional methods.
[0160] As understood herein, the methods of the instant invention
comprise administering the compositions of the invention to a
subject according to various regimens, i.e., in an amount and in a
manner and for a time sufficient to provide a clinically meaningful
benefit to the subject. Suitable administration regimens for use
with the instant invention may be determined by one of skill in the
art according to conventional methods. For example, it is
contemplated herein that an effective amount may be administered to
a subject as a single dose, a series of multiple doses administered
over a period of days, or a single dose followed by one or more
additional "boosting" doses thereafter. The term "dose" or "dosage"
as used herein refers to physically discrete units suitable for
administration to a subject, each dosage containing a predetermined
quantity of the active pharmaceutical ingredient calculated to
produce a desired response.
[0161] The administrative regimen, e.g., the quantity to be
administered, the number of treatments, and effective amount per
unit dose, etc. will depend on the judgment of the practitioner and
are peculiar to each subject. Factors to be considered in this
regard include physical and clinical state of the subject, route of
administration, intended goal of treatment, as well as the potency,
stability, and toxicity of the particular composition. As
understood by one of skill in the art, a "boosting dose" may
comprise the same dosage amount as the initial dosage, or a
different dosage amount. Indeed, when a series of doses are
administered in order to produce a desired response in the subject,
one of skill in the art will appreciate that in that case, an
"effective amount" may encompass more than one administered dosage
amount.
Diagnostics and Methods of Detecting Bacteria
[0162] Phage-based diagnostics and methods of detecting bacteria
are also currently being developed for clinical and industrial
applications. As discussed above, when a phage infects and
replicates within its bacterial host, that phage can increase in
titer from 10-100-fold in a single generation. The massive increase
in phage titer is a specific "signal" that can be easily monitored
by any number of techniques, including, e.g., classical phage titer
counts, quantitative real-time PCR probing the phage genome or
other reporter constructs, nucleic acid hybridization or other
molecular assays, and fluorescence or immunofluorescence assays
with labeled phage particles, etc. Phage diagnostics can
discriminate between live bacterial cells and the presence of dead
bacterial cells or cell debris since phages require a live host in
which to replicate. Thus, phage-based diagnostics and methods of
detecting bacteria are specific and powerful tools, but are
constrained by the same requirements as phage therapeutics. Thus,
in a particular embodiment, it is contemplated herein that the
methods of the instant invention may be employed to create phage
libraries for use in diagnostic applications, e.g., in a
personalized, library-to-diagnostic approach. The methods of the
instant invention provide new ways of harvesting diverse phages,
and phages against conditionally adapted bacterial hosts, including
host-adapted bacterial hosts, that will enable more powerful and
specific diagnostics.
[0163] In particular aspects, it is contemplated herein that
phage-based diagnostics may be used with clinical samples of a
biotic origin, e.g., samples of blood, sputum, puss, etc., and
industrial samples of an abiotic organ, e.g., swabs or otherwise
wet samples from industrial machinery, food, makeup, and other
pharmaceuticals, etc. Thus, identifying phages specific not only to
a bacterial host, but to a bacterial host grown under very specific
conditions, may be of particular importance to harvesting and
library construction for the purposes of diagnostics. It is
contemplated herein that the methods of the instant invention are
adaptable and may be modified to address specific industrial
diagnostic needs, e.g., employing phages identified according to
the methods of the instant invention in methods of detecting
bacterial contamination in various industrial applications (e.g.
food industry) comprising altering conditions and/or additives to
harvesting cultures, as needed by an end-user, without undue
experimentation.
Kits and Articles of Manufacture
[0164] It is contemplated herein that reagents for conditional
cultures disclosed herein, as well as compositions comprising
phages identified according to the methods of the invention, may be
provided to a user (e.g., a clinician treating a subject with a MDR
bacterial infection, or attempting to diagnose a bacterial
infection) in the form of a kit or other article of manufacture.
Kits comprising pharmaceuticals or other agents or items for
clinical use are familiar to one of skill in the art. Such kits may
take many forms; typically, they comprise one or more packaging
containers designed to safeguard the integrity and viability of the
contents during transit and/or storage. In a particular embodiment,
a kit of the instant invention may comprise one or more
compositions and may further comprise one or more additional
reagents or items for use therewith, e.g., buffers, diluents, etc.
as well as instructions or other information describing and/or
facilitating the administration of the kit contents. In various
embodiments, in addition to active pharmaceutical ingredients,
excipients, diluents, buffers, etc. the kits of the instant
invention may comprise various articles or medical devices made
from a variety of pharmaceutically acceptable materials or reagents
for facilitating treatment of a subject. These include, but are not
limited to, vials, syringes, IV bags, etc. Similar kits for
diagnostic or industrial purposed are contemplated herein, the
contents of which may be designed as proposed herein and determined
by one of skill in the art.
[0165] The compositions of the instant invention may be
administered to a patient alone, or in combination with one or more
pharmaceutical agents in any manner or dosing regimen, e.g.,
before, after, or concomitantly with one or more other
pharmaceutical or other therapeutic agent. Indeed, in a particular
embodiment, optimal therapy may comprise the integration of
bacteriophage therapy coupled to antibiotics and source control (if
possible) in parallel with optimization of the host immune
function. As understood by one of skill in the art, "source
control" refers to treating the infection directly at the source of
the infection in the subject, i.e., before the infection spreads
systemically. As discussed above, in addition to exploiting
bacteriophages for direct bacterial lysis, bacteriophages may act
synergistically with antibiotics in vivo, while potentiating
reversion of bacterial susceptibility to antibiotic classes.
[0166] Thus it is contemplated herein that administration of a
therapeutic phage cocktail may stress the emergent bacterial
strains such that the emergent bacterial strains regain sensitivity
to one or more drugs, e.g., an antibiotic to which it previously
demonstrated resistance. In addition, it is further contemplated
herein that phage cocktails of the instant invention may be
administered to a subject concurrently with one or more antibiotics
or other drugs to enhance overall therapeutic efficacy, e.g., to
produce a synergistic therapeutic effect. Thus, it is contemplated
herein that phages, and specifically the compositions of the
instant invention, may act synergistically with antibiotics, and/or
potentiate reversion of pathogen susceptibility to antibiotic
classes.
[0167] Although the invention herein has been described with
reference to particular embodiments, it is to be understood that
these embodiments, and examples provided herein, are merely
illustrative of the principles and applications of the present
invention. It is therefore to be understood that numerous
modifications can be made to the illustrative embodiments and
examples, and that other arrangements can be devised without
departing from the spirit and scope of the present invention as
defined by the appended claims. All patent applications, patents,
literature and references cited herein are hereby incorporated by
reference in their entirety.
EXAMPLES
Example 1: Using Conditional Cultures to Enhance the Isolation of
Bacteriophages with Therapeutic Potential Against Multidrug
Resistant ESKAPE Pathogens from the Peruvian Amazon
[0168] The experiments provided below describe efforts to establish
a diverse phage library against the ESKAPE pathogens, and
demonstrate that using conditional phage isolation cultures can
maximize the harvest of phages from environmental samples,
increasing the diversity (host range) of a final library.
Materials and Methods
MDR Strains
[0169] MDR clinical isolates of A. baumamnii (10 strains), K.
pneumoniae (15 strains), P. aeruginosa (20 strains) and S. aureus
(10 strains) were used to isolate phages from environmental water
samples. These clinical isolates were collected in Iquitos, Peru,
between 2011 and 2017 and each had a distinct isolation date and/or
AST profile.
Phage Isolation and Purification
[0170] Environmental sewage water was collected in and around the
Peruvian Amazon city of Iquitos. 3% w/v TSB-powder was directly
mixed with 300 mL of the raw sewage and inoculated with 1 mL of
each MDR strain in exponential phase, with 5 strains max per 300 mL
to limit competition, and incubated at 37.degree. C. shaking
overnight. Following growth, 1.5 mL of the overnight
bacteria-sewage culture was centrifuged at 12000 rpm for 1 min, and
the phage-rich supernatant was filtrated with 0.22 .mu.m filters to
remove remaining bacteria. Serial dilutions of each sterile
supernatant was then spotted (10 .mu.L) onto lawns of each
individual bacteria (TSB agar), and incubated at 37.degree. C.
overnight. Resulting single phage plaques were harvested by
removing the plaque and agar plug with a Pasteur pipette and
incubating the plugs in 300 .mu.L of PBS for 60 min. The
resuspended phages were then sterilized using 0.22 .mu.m Spin-X
centrifuge filters. The species-specific host range of all the
isolated phages (1:5 dilution) were identified against the original
bacterial strains used for phage isolation and additional MDR
strains not used for isolation. Total strains for host-range
analysis: 15 A. baumannii, 20 K. pneumoniae, 20 P. aeruginosa, and
20 S. aureus.
Conditional Culture Phage Isolation
[0171] Phage isolation was repeated with 10 MDR K. pneumoniae
strains using the same sample of environmental water split into
traditional TSB isolation cultures and TSB cultures containing 7.5%
Fetal Bovine Serum (FBS). FBS is known to alter gene expression in
bacterial pathogens. Phage isolation from the same water source
using these two conditions was then compared, and the host ranges
of the isolated phages was analyzed against the same 20 K.
pneumoniae strains as before.
Results
Phage Harvesting Using Traditional Isolation
[0172] In total, 8 phages were found against 8 of the 10 A.
baumannii (AB) strains, from 1 water source; 33 phages were found
against 14 of the 15 K. pneumoniae (KP) strains, from 2 water
sources; 17 phages were found against 16 of the 20 strains of P.
aeruginosa (PA), from 2 water sources; 5 phages were found against
5 of the 10 strains of S. aureus (SA) from 1 water source.
[0173] In addition, 63% (5/8) of the AB phages were isolation-host
specific and 37% (3/8) were not, infecting between 4 and 7 of the
AB strains including their host (FIG. 1). 64% (21/33) of the KP
phages were isolation-host specific and 36% (12/33) were not,
infecting between 2 and 7 of the KP strains including their host
(FIG. 2). All of the PA phages were not isolation-host specific;
29% (5/17) of the PA phages infected between 3 and 5 PA strains and
71;% (12/17) infected more than 10 strains (FIG. 3). Similarly, all
of the SA phages were not isolation-host specific and infected all
16 SA strains (FIG. 4).
Conditional Phage Harvesting
[0174] Data indicate that 6 of the 10 KP strains yielded phages
with identical host ranges in both conditions: KP01, KP04, KP06,
KP08, KP11 and KP12. In addition, 4 of the 10 KP strains yielded
phages with conditional dependence: KP03, KP07, KP09 and KP13.
[0175] Notably, as determined by host range, KP03, KP09 and KP13
all yielded phages in the presence of FBS that were not found
without FBS. Conversely, KP03 and KP07 yielded phages in the
absence of FBS that were not found in the presence of FBS. In
addition, 5 strains showed conditionally-dependent phage titers:
KP03, KP04, KP07, and KP12 showed an apparent increase in phage
titer with FBS, while KP06 showed an apparent increase without FBS.
Importantly, with all phages found, the isolation condition was not
required for infectivity.
Discussion
[0176] Data from conditional phage harvesting indicate that varying
isolation conditions changes the cohort of the resulting isolated
phages from the same water sample. The single sample of
environmental water was dived into two portions: (1) one portion
was used for traditional phage harvesting in classical TSB culture
media, and (2) a second portion was used under identical harvesting
conditions, except the second culture also contained 7.5% FBS. The
addition of FBS to the second isolation culture was the only
difference and both cultures were used to isolate phages against K.
pneumoniae. Referring to FIG. 5, each row in FIG. 5 corresponds to
a single K. pneumoniae host strain, and each phage head (hexagon)
in the row represents an individual phage isolated against the
corresponding strain, +/-FBS (Columns). A critical feature for
phage therapeutics is a phage's host range. Phages with different
host ranges are functionally different phages, regardless of how
similar their genomes are. The number in each phage head in FIG. 5
is the number of different K. pneumoniae strains that phage can
infect, out of a total of 20 K. pneumoniae strains tested. The "+"
next to the number indicates that the titer of that isolated phage
was increased under that condition with respect to the other
condition.
[0177] An example of interpreting FIG. 5 is as follows: Against
KP01 was found 2 total phages, 1 was found without FBS and 1 was
found in the presence of FBS (each column only has 1 phage head).
Each phage can only infect KP01 (the number inside each phage head,
in each column, is "1"). Thus, these two phages against KP01 are
functionally identical. Against KP03 was found 4 total phages (4
phage heads), 2 were found without FBS and 2 were found with FBS.
With regard to the two phages with a total host range of 4 (there
is a 4 in the phage head), 1 found without FBS and 1 found with
FBS, these two phages are functionally the same and have identical
host ranges. With respect to the phage with a host range of 3
(there is a 3 inside the phage head), this phage is distinct from
the phages with a total host range of 4, and critically was not
found in the presence of FBS. Conversely, the phage that only
infects KP03 and has a host range of 1 (there is a 1 in the phage
head), this phage was found in the presence of FBS and was not
found in the absence of FBS.
[0178] Importantly, all the phages isolated here, in either
condition, were capable of still infecting its host in the other
condition, once the phage had been isolated and purified.
Additionally, with KP07 for example, the phage with a host range of
4 was isolated a total of twice, as was the KP06 phage with a total
host range of 6. Thus such conditionally isolated phages can be
found reliably, and NOT in the other condition.
[0179] Comparing phage isolation against KP01 and KP03: [0180] (1).
some conditional changes do not alter the cohort of isolated phages
against a strain. [0181] (2). +/-FBS changed the cohort of phages
isolated against KP03 from the exact same water source, with a
phage only found in the absence of FBS and a phage only found in
the presence of FBS, because of bacterial gene expression and
physiological changes between these two conditions. [0182] (3).
There are potentially many more phages in the wild that cannot be
isolated with traditional cultures and/or there is a competition
between phages in isolation cultures. [0183] (4). Varying more
features of an isolation culture, according to the specifications
of the instant invention here, could potentially yield many more
diverse phages and maximize phage isolation. Similarly, if one
alters in vivo culture conditions to mirror the host environment,
it will be possible to isolate phages better suited to infecting
bacteria as they grow during a bona fide infection. Isolating
phages using conditional cultures will maximize phage harvesting
and phage diversity, with phage diversity being essential for
personalized therapeutic cocktail formulation, and having diverse
phages will greatly assist other phage therapeutic modalities as
well.
Conclusions
[0184] Peruvian water sources yielded phages to all pathogens,
allowing for the expansion of existing phage libraries against the
ESKAPEs. Generally, AB and KP phages isolated here have narrower
host ranges than do PA and SA phages. Conditional cultures using
FBS can effect phage-yield with respect to the identities of the
phages isolated, based on host range, and the amount of the yield
(titer) of some phages. Here, +/-FBS allows for the isolation of
more diverse phages, even from the exact same environmental water
sample. Also, +/-FBS also resulted in both the loss and the gain of
isolated phages from different strains, suggesting that conditional
cultures can alter bacterial physiology and cause a loss or a gain
in phage infectivity.
[0185] Taken together, the results suggest that conditional
cultures may be used to maximize phage isolation from environmental
water sources, resulting in the construction of a more diverse
phage library. Moreover, these data suggest that it is possible to
use conditional cultures to possibly mirror host-like conditions to
force bacterial gene expression and the available receptor
repertoire to be more host-adapted-like, and possibly find phages
that are better therapeutic candidates. Indeed, even if attempts to
mirror in vivo conditions in vitro are never completely successful,
data indicate that it is possible to demonstrably alter phage
isolation culture conditions so as to enhance (and even possibly
maximize) phage isolation from even the exact same environmental
water sample and thus build a larger and more diverse and more
useful phage library.
Example 2: Conditional Phage Harvesting in the Presence of Host
Blood Products
[0186] Subsequent to the collection of data described in Example 1,
the experimental methods described therein were repeated in another
set of experiments with different strains of the same bacterial
species and different water samples. Specifically, the method
employed was exactly the same as provided above, with the exception
that additional cultures containing TSB with 5% sheep blood were
also used, in addition to TSB alone and TSB with 7.5% FBS. In
addition, some cultures were grown at room temperature.
[0187] Details of the experiments performed, and the results
obtained in these studies are presented in FIGS. 6, 7, 8A, 8B, 9A,
9B, 10 A and 10B. Data in these figures illustrate in numerous
bacterial strains and species that the simple addition of FBS or
sheep's blood to harvesting cultures not only diversifies phage
harvesting across the set of harvesting cultures, but also
indicates that there are phages that are conditionally isolated
(when scored by host range, arguably the most important feature of
a phage with respect to its therapeutic potential), i.e., there are
phages present in a single sample that can be isolated in the
presence of FBS or sheep's blood, that cannot be isolated with
media-only traditional cultures, and vice versa. These results
illustrate that although these phages are present across the set of
cultures via the homogenous aliquoted sample, the conditional
cultures produce different "winners" and "losers" from each
culture's interphage competition, i.e., "different phage
competition outcomes." The exact mechanism as to how these cultures
are affecting the bacteria and/or modulating the phage competition
outcome is not known, but since the method works in multiple
different species, and since in each species the mechanism may
vary, the method is nevertheless very robust and likely involves
bacterial gene expression differences across the culture set.
Future experiments using concentrated mouse organ homogenates
(discussed below in Example 3) may improve the ability of
conditional culture sets to find even more phages.
[0188] Data presented herein indicate that not only are there
phages that show conditionally dependent isolation, but there are
phages that show conditionally independent and conditionally
dependent infection of the same host. For example, FIG. 6
harvesting data with bacterial strain WIQ0239 illustrates that a
phage was found against this strain in the presence of sheep's
blood, and that this phage cannot infect the WIQ0239 host without
sheep's blood present. Also, FIG. 6 harvesting data with bacterial
strain WIQ0289 illustrates that there was a phage found that was
capable of infecting the WIQ0289 host in traditional culture media
and under traditional conditions, but cannot infect when either FBS
or sheep's blood is added to the culture. This remarkable result
indicates that there are phages in the wild that cannot infect
their host strain in the presence of biological materials like FBS
and sheep's blood, and there are some phages that are incapable of
infecting their host strains in the absence of such biological
materials.
[0189] The data in FIG. 6 concerning phage against WIQ0289 isolated
in traditional media cultures that cannot infect in the presence of
FBS or sheep's blood indicate that blood, or a component of blood,
blocks phage infectivity in these cultures either directly or by
altering bacterial gene expression away from allowing the phage to
infect. Thus, it is hypothesized herein that such a phage may show
promise in vitro as a potential therapeutic phage, but may fail
when used in a host because of serum or blood effects (the
mechanism of which may involve direct antagonism at the phage
level, or may alter gene expression in the bacteria, or both). This
result demonstrates the inherent insufficiency of traditional phage
harvesting and the utility of the methods of the instant
invention.
[0190] Taken together, these data indicate that 1) harvesting
cultures contain numerous phages which compete, and that
competition can be modulated via changing conditions, allowing for
increased phage harvesting from a signal environmental sample when
such harvesting is iterated across a set of multiple conditional
cultures, and 2) altering bacterial culture conditions, including
the addition of host material such as blood or serum, likely
changes bacterial gene expression and surface receptor identities
effecting phage infectivity. Indeed, bacteria grown under specific
culture conditions may not express receptors that are present in
other conditions or in vivo, and similarly bacteria in vitro may
express surface receptors that are not expressed at all in vivo.
Unfortunately, such receptors may act as "decoys" in conventional
methods of phage harvesting by selecting for phages that can infect
and kill a bacterial strain in vitro, and/or only under certain
specific conditions in vitro, and which actually have little
therapeutic efficacy on the same bacterial strain in vivo.
[0191] Consistent with the data in FIG. 6 concerning K. pneumoniae
phages, the data in FIGS. 7, 8A, 8B, 9A and 9B illustrate that
conditionally dependent phage isolation and conditionally dependent
phage infection are also seen in P. aeruginosa, again when phage
identity is judged by bacterial host range. For example, in FIG.
8B, phages against strain P. aeruginosa NSI1489 show conditionally
dependent isolation, e.g., the phage isolated from media alone is
not found in harvesting cultures containing FBS, and vice versa,
and the NSI1489 phages also all show conditionally dependent
infection, e.g., each phage in that row infects a smaller set of
host strains in the presence of blood (bottom ratio), as compared
to media alone (top ratio) and media with FBS (middle ratio).
Importantly, for example, if one were to assess these phages in
vitro in media only cultures for their ability to infect and kill a
clinical isolate of interest causing bacteremia in a patient, and
thus be used in a phage therapeutic against said bacteremia, the
media only results would not predict the performance or accurate
host-range of these phages as they infect very differently in the
presence of blood. These data suggest that this is a significant
weakness of classical phage harvesting and phage host-range
determination.
[0192] Additionally, FIGS. 7, 8A, 8B, 9A and 9B reveal that there
are very different repertoires of phages in the different water
samples. For example, the data in FIGS. 7 and 8A show that with
those two different water sources, there were no P. aeruginosa
phages present that could infect the targeted P. aeniginosa strains
in the presence of FBS. By contrast, FIG. 8B demonstrates that the
water source contained no P. aeruginosa phages capable of infecting
in the presence of sheep blood. Thus, not only do these results
demonstrate that conditional phage isolation and conditional phage
infection are not phenomena unique to K. pneumoniae, as they occur
in P. aeruginosa as well, indicating that these phenomena are
likely generalizable to all bacterial pathogens, but these data
also demonstrate the importance of diversifying the environmental
sources used for phage isolation.
[0193] The data presented in FIGS. 10A and 10B further demonstrates
the generalizable phenomena of conditional phage isolation and
conditional phage infection with respect to phages against A.
baumannii. In FIG. 10A, phages against strain WIQ0105 show
conditional phage isolation and conditional phage infection.
[0194] On the whole, the data presented in FIGS. 6, 7, 8A, 8B, 9A,
9B, 10A and 10B demonstrate that conditionally dependent phage
isolation and conditionally dependent phage infection can be seen
in K. pneumoniae, P. aeruginosa, and A. baumannii, indicating that
conditional effects on phage infection are broadly
generalizable.
[0195] One can argue that the addition of blood in the cultures
creates an environment for the bacteria that is more "host-like."
Interestingly, the data indicate that some phages have
significantly smaller host ranges in the presence of blood. Thus,
the presence of blood in the culture apparently has an impact on
the phages, the bacteria, or both, which can change phage
infectivity. A very reasonable explanation is that some of the host
bacteria change gene expression in the presence of blood and that
changes phage infection dynamics.
[0196] In addition, data indicate that with a particular water
sample, phages could not be isolated against any of the bacterial
host strains in the presence of serum, but it was possible in
media. Presumably none of the media harvested phages will infect
under certain conditions in the presence of host material like
serum. Thus, these data suggest that traditional studies using
conventional media only infection and harvesting conditions may
lead to over-predictions with regard to phage infectivity in
vivo.
[0197] Significantly, taken together, the data support the concept
disclosed herein that aliquoting a sample comprising phages across
a set of cultures that conditionally vary, and thereby modulating
phage competition outcomes across the set, optimizes and enhances
phage harvesting from a single environmental source and allows the
recovery of phages from that sample that cannot be found otherwise,
likely because the same bacteria strain is expressing different
surface features across the set of conditional harvesting cultures.
Thus, in effect, the bacteria are literally different across the
set of cultures with respect to phage harvesting.
Example 3: Experiments Using Concentrated Mouse Organ
Homogenates
[0198] Various possible culture features and additives may be
employed in the methods of the invention. For example, future
experiments involving creating different culture conditions
comprising using various concentrated mouse organ homogenates are
contemplated herein. These experiments may be performed as
follows.
Materials and Methods:
[0199] Homogenates of various mouse tissues have been prepared.
Specifically, livers, spleens, kidneys, hearts, and brains from
uninfected BALB/c mice have been prepared by pooling said organs
from over 50 mice and homogenizing the material into 50 ml PBS, pH
7.2.
[0200] Whole mouse organs, including liver, brain, heart, spleen,
kidney, muscle tissue, and bone, homogenized together or separately
in PBS, could be added in various concentrations (e.g. 5%-25%) to
phage harvesting cultures. These homogenates will contain very
complex mixtures of host-derived materials in solution as well as
insoluble materials that could provide host-specific surfaces. It
is contemplated herein that using the general methods described
above for phage harvesting, but using harvesting cultures that
contain these kinds of mouse organ homogenates, will be superior to
FBS and/or blood at mimicking the host environment and producing
host-adapted bacteria in said harvesting cultures.
[0201] In a particular embodiment, it is proposed that these
homogenates may be utilized in additional future experiments
according to the methods of the invention as proposed below. Phage
harvesting according to the methods of the instant invention,
specifically as performed and data presented in FIGS. 6, 7, 8A, 8B,
9A, 9B, 10A, and 10B, will be repeated with fresh environmental
water samples but now with a fourth condition. Thus, in addition to
media, media+7.5% FBS, and media+5% sheep blood, a harvesting
culture comprising media+5% of the moue organ homogenate described
above will be used. Such a strategy using all four conditions will
allow for comparisons across all the conditions, looking for
changes in or enhancements of conditional phage isolation and/or
conditional phage infection in the presence of mouse organ
homogenates. It is also contemplated herein that a fifth
conditional harvesting culture may also be generated that contains
all of the additives, e.g. FBS, sheep blood, as well as mouse organ
homogenates. Such a culture may prove to be superior to all
previously tested conditions.
* * * * *