U.S. patent application number 16/630174 was filed with the patent office on 2021-01-28 for potentiation of antibiotic effect.
This patent application is currently assigned to The University of North Carolina at Chapel Hill. The applicant listed for this patent is THE UNIVERSITY OF NORTH CAROLINA AT CHAPELl HILL. Invention is credited to Brian CONLON, Sarah CONLON, Lauren RADLINSKI.
Application Number | 20210023101 16/630174 |
Document ID | / |
Family ID | 1000005190186 |
Filed Date | 2021-01-28 |
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United States Patent
Application |
20210023101 |
Kind Code |
A1 |
CONLON; Brian ; et
al. |
January 28, 2021 |
POTENTIATION OF ANTIBIOTIC EFFECT
Abstract
The present invention relates to compositions, kits and methods
using combinations of surfactants or cell wall-hydrolyzing enzymes
with antibiotics to potentiate the antibiotic effect against
bacterial infections. In some embodiments, the surfactant is a
pore-forming biosurfactant such as rhamnolipids and the antibiotic
is an aminoglycoside antibiotic such as tobramycin.
Inventors: |
CONLON; Brian; (Durham,
NC) ; CONLON; Sarah; (Durham, NC) ; RADLINSKI;
Lauren; (Carrboro, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF NORTH CAROLINA AT CHAPELl HILL |
Chapel Hill |
NC |
US |
|
|
Assignee: |
The University of North Carolina at
Chapel Hill
Chapel Hill
NC
|
Family ID: |
1000005190186 |
Appl. No.: |
16/630174 |
Filed: |
July 19, 2018 |
PCT Filed: |
July 19, 2018 |
PCT NO: |
PCT/US2018/042800 |
371 Date: |
January 10, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62534450 |
Jul 19, 2017 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/702 20130101;
A61K 47/26 20130101; A61P 31/04 20180101 |
International
Class: |
A61K 31/702 20060101
A61K031/702; A61K 47/26 20060101 A61K047/26; A61P 31/04 20060101
A61P031/04 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under
AI125501 and AI137273 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A composition comprising a surfactant and an aminoglycoside
antibiotic.
2. The composition of claim 1, wherein the surfactant is a
pore-forming biosurfactant.
3. The composition of claim 2, wherein the pore-forming
biosurfaetant is a rhamnolipid.
4. The composition of claim 3, wherein the rhamnolipid is from
Pseudomonas aeruginosa.
5. The composition of claim 3, wherein the rhamnolipid is a
mono-rhamnolipid, a di-rhamnolipid, or a combination thereof.
6. The composition of claim 2, wherein the pore-forming
biosurfactant is a xylolipid.
7. The composition of claim 1, wherein the surfactant and the
aminoglycoside antibiotic are present in synergistic amounts.
8. The composition of claim 1, wherein the aminoglycoside
antibiotic is selected from the group consisting of streptomycin,
kanamycin A, amikacin, tobramycin, dibekacin, gentamicin,
sisomicin, netilmicin, neomycin B, neomycin C, and neomycin E
(paromornycin).
9. The composition of claim 8, wherein the aminoglycoside
antibiotic is tobramycin.
10. A kit comprising a surfactant and an aminoglyeoside
antibiotic.
11-21. (canceled)
22. A method of treating a bacterial infection in a subject in need
thereof, comprising administering to the subject a therapeutically
effective amount of a surfactant and an aminoglycoside antibiotic,
wherein the bacterial infection, is treated.
23-24. (canceled)
25. The method of claim 21, wherein the bacteria is antibiotic
resistant or antibiotic tolerant.
26. The method of claim 21, wherein the surfactant decreases the
minimum inhibitory concentration of the aminoglycoside
antibiotic.
27. The method of claim 21, wherein the bacteria is a gram-negative
bacteria.
28. The method of claim 21, wherein the bacteria is a gram-positive
bacteria.
29. The method of claim 28, wherein the bacteria is Staphylococcus
aureus.
30. The method of claim 29, wherein the bacteria is
methicillin-resistant Staphylococcus aureus.
31-32. (canceled)
33. The method of claim 21, wherein the surfactant and the
aminoglycoside antibiotic are administered in synergistic
amounts.
34. The method of claim 21, wherein the aminoglycoside antibiotic
is selected from the group consisting of streptomycin, kanamycin A,
amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin,
neomycin. B, neomycin C, and neomycin (paromornycin).
35. (canceled)
36. The method of claim 21, wherein he surfactant is a pore-forming
biosurfactant,
37-40. (canceled)
Description
STATEMENT OF PRIORITY
[0001] This application is a 35 U.S.C. .sctn. 371 national phase
application of PCT Application PCT/US2018/042800 filed Jul. 19,
2018, which claims the benefit of U.S. Provisional Application Ser.
No. 62/534,450, filed Jul. 19, 2017, the entire contents of each of
which are incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to compositions, kits and
methods using combinations of surfactants or cell wall-hydrolyzing
enzymes with antibiotics to potentiate the antibiotic effect
against bacterial infections.
BACKGROUND OF THE INVENTION
[0004] Staphylococcus aureus is a major human pathogen responsible
for numerous chronic and relapsing infections. These infections
often fail to respond to antibiotic treatment, even in the apparent
absence of antibiotic resistance. Additionally, antibiotic
resistance of microorganisms such as S. aureus is a growing dilemma
worldwide. The overuse of currently available antibiotics has
contributed to this issue. The development of new antibiotics and
alternative methods of treating infections is needed.
[0005] There remains a need for compounds and combinations of
compounds that can increase the sensitivity of bacteria to
antibiotics and treat bacterial infections.
SUMMARY OF THE INVENTION
[0006] The present invention is based, in part, on the development
of effective combinations of surfactants or cell wall-hydrolyzing
enzymes and other antibiotics that enhance the activity of the
other antibiotics and increase the antibiotic sensitivity of
bacteria.
[0007] Accordingly, one aspect of the invention relates to a
composition comprising a surfactant and an aminoglycoside
antibiotic.
[0008] Another aspect of the invention relates to a kit comprising
a surfactant and an aminoglycoside antibiotic.
[0009] Another aspect of the invention relates to a method of
increasing the sensitivity of a bacteria to an aminoglycoside
antibiotic, comprising contacting the bacteria with an effective
amount of a surfactant and the aminoglycoside antibiotic.
[0010] Another aspect of the invention relates to a method of
treating a bacterial infection in a subject in need thereof,
comprising administering to the subject a therapeutically effective
amount of a surfactant and the aminoglycoside antibiotic.
[0011] Another aspect of the invention relates to a method of
reducing the risk of recurrence of a bacterial infection in a
subject in need thereof, comprising administering to the subject
having a bacterial infection a therapeutically effective amount of
a surfactant and the aminoglycoside antibiotic.
[0012] Another aspect of the invention relates to a method of
disrupting a biofilm, comprising contacting the biofilm with an
effective amount of a surfactant and the aminoglycoside
antibiotic.
[0013] Another aspect of the invention relates to a composition
comprising a cell wall-hydrolyzing enzyme and a cell wall synthesis
inhibitor glycopeptide antibiotic.
[0014] Another aspect of the invention relates to a kit comprising
a cell wall-hydrolyzing enzyme and a cell wall synthesis inhibitor
glycopeptide antibiotic.
[0015] Another aspect of the invention relates to a method of
increasing the sensitivity of a bacteria to a cell wall synthesis
inhibitor, comprising contacting the bacteria with an effective
amount of a cell wall-hydrolyzing enzyme and the cell wall
synthesis inhibitor glycopeptide antibiotic.
[0016] Another aspect of the invention relates to a method of
treating a bacterial infection in a subject in need thereof,
comprising administering to the subject a therapeutically effective
amount of a cell wall-hydrolyzing enzyme and a cell wall synthesis
inhibitor glycopeptide antibiotic.
[0017] Another aspect of the invention relates to a method of
reducing the risk of recurrence of a bacterial infection in a
subject in need thereof, comprising administering to the subject
having a bacterial infection a therapeutically effective amount of
a cell wall-hydrolyzing enzyme and a cell wall synthesis inhibitor
glycopeptide antibiotic.
[0018] Another aspect of the invention relates to a method of
disrupting a biofilm, comprising contacting the biofilm with an
effective amount of a cell wall-hydrolyzing enzyme and a cell wall
synthesis inhibitor glycopeptide antibiotic.
[0019] These and other aspects of the invention are set forth in
more detail in the description of the invention below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A-1D show P. aeruginosa supernatant alters S. aureus
antibiotic susceptibility. S. aureus strain HG003 was grown to
mid-exponential phase and pre-treated with sterile supernatant from
either P. aeruginosa PAO1, PA14 or S. aureus HG003 for 30 min.
Cultures were then challenged with the C.sub.max of (A)
ciprofloxacin (B) oxacillin (C) vancomycin or (D), tobramycin. At
indicated times, an aliquot from each culture was washed and plated
to enumerate survivors. All experiments were performed in
biological triplicate. Error bars represent mean.+-.sd.
[0021] FIGS. 2A-2D show P. aeruginosa secondary metabolites inhibit
S. aureus aerobic respiration resulting in a drop in intracellular
ATP. (A) S. aureus strain HG003 harboring plasmid PpflB::gfp was
grown to mid-exponential phase and treated with P. aeruginosa PAO1
supernatant, PA14 supernatant or S. aureus HG003 supernatant for 30
min. OD600 and gfp expression levels were determined using a Biotek
Synergy HI microplate reader. (B) Intracellular ATP was measured
after 1.5 h incubation with supernatant. **p<0.001,
***p<0.0005 (Student's t-test). (C) S. aureus strain HG003 was
grown to mid-exponential phase in MHB media and pre-treated with
sterile supernatants from P. aeruginosa strains PA14 wild-type or
its isogenic mutants or (D) clinically relevant concentrations of
HQNO, pyocyanin (PYO) or sodium cyanide (NaCN) for 30 min prior to
antibiotic challenge. At indicated times, an aliquot was washed and
plated to enumerate survivors. All experiments were performed in
biological triplicate. Error bars represent mean.+-.sd.
[0022] FIGS. 3A-3C show P. aeruginosa supernatant potentiates
killing by vancomycin via the LasA endopeptidase. S. aureus HG003
was grown to mid-exponential phase and exposed to sterile
supernatants for 30 min prior to addition of vancomycin 50
.mu.g/ml. Where indicated, PA14 supernatant was heat inactivated
(PA14 HI) at 95.degree. C. for 10 min (A, C) At indicated times, an
aliquot was removed, washed and plated to enumerate survivors or
(B) 100 .mu.l cells were added to a 96-well plate and lysis was
measured at OD.sub.600 every hr for 16 h. All experiments were
performed in biological triplicate. Error bars represent
mean.+-.sd.
[0023] FIGS. 4A-4D show P. aeruginosa rhamnolipids potentiate
aminoglycoside uptake and cell death in S. aureus. S. aureus HG003
was grown to mid-exponential phase and exposed to (A, B) sterile
supernatants from P. aeruginosa or S. aureus or (C) exogenous
addition of rhamnolipids 10-50 .mu.g/ml before addition of
tobramycin 58 .mu.g/ml. At indicated times, an aliquot was washed
and plated to enumerate survivors. (D) Texas Red-conjugated
tobramycin was added to S. aureus cultures with or without 30
.mu.g/ml rhamnolipids. Following 1 h, Texas Red-tobramycin uptake
was measured by flow cytometry. Experiments were performed in
biological triplicate. Error bars represent mean.+-.sd.
[0024] FIGS. 5A-5F show clinical P. aeruginosa isolates produce a
wide range of antistaphylococcal factors and alter antibiotic
tolerance in S. aureus. Antistaphylococcal factors present in the
supernatant of P. aeruginosa CF isolates (blue) or burn isolates
(green) were quantified as follows. (A) HQNO was measured by
PpflB::gfp reporter induction. (B) LasA was quantified by Western
blot and the ability of each supernatant to lyse heat-killed S.
aureus HG003 cells after 2 h. (C) Rhamnolipid production was
measured by a drop-collapse assay. (D-F) S. aureus HG003 was grown
to mid-exponential phase and exposed to sterile supernatants from
clinical isolates or the control, S. aureus HG003 (red) for 30 min
prior to addition of (D) ciprofloxacin (E) vancomycin or (F)
tobramycin. An aliquot was removed after 24 h, washed and plated to
enumerate survivors. All experiments were performed in biological
triplicate, with the exception of (E) which was performed in
biological duplicate. Error bars represent mean.+-.sd.
[0025] FIG. 6 shows P. aeruginosa mediated alteration of S. aureus
antibiotic susceptibility. P. aeruginosa exoproducts pyocyanin
(PYO), 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), and hydrogen
cyanide (HCN) inhibit S. aureus electron transport, leading to
collapse of proton-motive force (PMF) and inhibition of the
F.sub.1F.sub.0 ATPase leading to a decrease in S. aureus antibiotic
susceptibility. Conversely, P. aeruginosa rhamnolipids (RL)
intercalate into the plasma membrane forming pores that permit
aminoglycoside entry into the cell in a PMF-independent manner.
Finally, P. aeruginosa endopeptidase LasA cleaves pentaglycine
crosslinks between peptidoglycan molecules of the cell wall,
increasing vancomycin-mediated lysis of S. aureus.
[0026] FIGS. 7A-7E show S. aureus strain HG003 was grown to
mid-exponential phase in MHB media and pre-treated with (A) sterile
supernatants from P. aeruginosa strains PA14 wild-type or its
isogenic mutants or (C-E) clinically relevant concentrations of
HQNO, pyocyanin (PYO) or sodium cyanide (NaCN) for 30 min prior to
antibiotic challenge. (B) HG003 was grown to mid-exponential phase
in TSB+100 mM MOPS in an anaerobic chamber and pre-treated with
sterile supernatants from HG003 or PA14 for 30 min before addition
of ciprofloxacin. At indicated times, an aliquot was washed and
plated to enumerate survivors. All experiments were performed in
biological triplicate. Error bars represent mean.+-.sd.
[0027] FIGS. 8A-8B show S. aureus strain HG003 was grown to
mid-exponential phase in MHB media and pre-treated with (A) sterile
supernatants from P. aeruginosa strains PA14, heat-inactivated PA14
(PA14 HI), or (B) exogenous rhamnolipids 10-50 .mu.g/ml before
addition of tobramycin at 58 .mu.g/ml. At indicated times, an
aliquot was washed and plated to enumerate survivors. All
experiments were performed in biological triplicate. Error bars
represent mean.+-.sd.
[0028] FIG. 9 shows biofilm killing using tobramycin and
rhamnolipids.
[0029] FIG. 10 shows anaerobic killing using tobramycin and
rhamnolipids.
[0030] FIG. 11 shows rhamnolipids at 30 .mu.g/ml potentiates
killing of S. aureus HG003 by tobramycin.
[0031] FIG. 12 shows rhamnolipids at 50 .mu.g/ml dramatically
reduces the MIC of tobramycin against a variety of tobramycin
resistant clinical isolates taken from cystic fibrosis patients'
lungs.
[0032] FIG. 13 shows rhamnolipids sensitize low energy (arsenate
treated) S. aureus to tobramycin killing.
[0033] FIG. 14 shows rhamnolipids sensitize S. aureus to tobramycin
in the presence of CCCP which collapses membrane potential and
classical tobramycin uptake.
[0034] FIG. 15 shows protein synthesis inhibition inhibits
tobramycin killing but does not inhibit tobramycin mediated killing
in the presence of rhamnolipids.
[0035] FIG. 16 shows sub-culturing S. aureus in increasing
concentrations of tobramycin results in swift selection for highly
resistant isolates. However, no selection for highly resistant
isolates occurs in the presence of rhamnolipids.
[0036] FIG. 17 shows small colony variant (SCV) of S. aureus ,
HG003 menD::erm, is resistant to tobramycin but in the presence of
rhamnolipids can be killed by tobramycin.
[0037] FIG. 18 shows rhamnolipids cause a 511-fold collapse in the
MIC of SCV strain to tobramycin.
[0038] FIGS. 19A-19B show other membrane permeabilizing molecules
also synergize with tobramycin. (A) Propidium iodide uptake
experiments confirmed that palmitoleic acid (Palm50) and glycerol
monolaureate (GML) permeabilize S. aureus . (B) Killing experiments
with tobramycin showed significant potentiation of tobramycin
killing over 24 hours.
[0039] FIG. 20 shows the effect of different rhamnolipids on
potentiation of tobramycin.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention will now be described in more detail
with reference to the accompanying drawings, in which preferred
embodiments of the invention are shown. This invention may,
however, be embodied in different fowls and should not be construed
as limited to the embodiments set forth herein. Rather, these
embodiments are provided so that this disclosure will be thorough
and complete, and will fully convey the scope of the invention to
those skilled in the art.
[0041] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
skill in the art to which this invention belongs. The terminology
used in the description of the invention herein is for the purpose
of describing particular embodiments only and is not intended to be
limiting of the invention. All publications, patent applications,
patents, patent publications and other references cited herein are
incorporated by reference in their entireties for the teachings
relevant to the sentence and/or paragraph in which the reference is
presented.
[0042] Amino acids are represented herein in the manner recommended
by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino
acids) by either the one-letter code, or the three letter code,
both in accordance with 37 C.F.R. .sctn. 1.822 and established
usage.
[0043] As used in the description of the invention and the appended
claims, the singular forms "a," "an," and "the" are intended to
include the plural forms as well, unless the context clearly
indicates otherwise.
[0044] Also as used herein, "and/or" refers to and encompasses any
and all possible combinations of one or more of the associated
listed items, as well as the lack of combinations when interpreted
in the alternative ("or").
[0045] The term "about," as used herein when referring to a
measurable value such as an amount of polypeptide, dose, time,
temperature, enzymatic activity or other biological activity and
the like, is meant to encompass variations of .+-.10%, .+-.5%,
.+-.1%, .+-.0.5%, or even .+-.0.1% of the specified amount.
[0046] The transitional phrase "consisting essentially of" means
that the scope of a claim is to be interpreted to encompass the
specified materials or steps recited in the claim, and those that
do not materially affect the basic and novel characteristics of the
claimed invention.
[0047] The term "modulate," "modulates," or "modulation" refers to
enhancement (e.g., an increase) or inhibition (e.g., a decrease) in
the specified level or activity.
[0048] The term "enhance" or "increase" refers to an increase in
the specified parameter of at least about 1.25-fold, 1.5-fold,
2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold,
twelve-fold, or even fifteen-fold.
[0049] The term "inhibit" or "reduce" or grammatical variations
thereof as used herein refers to a decrease or diminishment in the
specified level or activity of at least about 15%, 25%, 35%, 40%,
50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments,
the inhibition or reduction results in little or essentially no
detectible activity (at most, an insignificant amount, e.g., less
than about 10% or even 5%).
[0050] A "therapeutically effective" amount as used herein is an
amount that provides some improvement or benefit to the subject.
Alternatively stated, a "therapeutically effective" amount is an
amount that will provide some alleviation, mitigation, or decrease
in at least one clinical symptom in the subject. Those skilled in
the art will appreciate that the therapeutic effects need not be
complete or curative, as long as some benefit is provided to the
subject.
[0051] By the terms "treat," "treating," or "treatment of," it is
intended that the severity of the subject's condition is reduced or
at least partially improved or modified and that some alleviation,
mitigation or decrease in at least one clinical symptom is
achieved.
[0052] A "synergistic" effect, as used herein, is an effect that is
greater than additive when two molecules are administered to a
subject simultaneously or sequentially.
[0053] The term "therapeutic index," as used herein, refers to the
ratio of the dose of drug that causes adverse effects at an
incidence/severity not compatible with the targeted indication
(e.g., toxic dose in 50% of subjects, TD50) to the dose that leads
to the desired pharmacological effect (e.g., efficacious dose in
50% of subjects, ED50). A widening of the therapeutic index refers
to an increase in the difference between the toxic and therapeutic
dose.
[0054] The term "antibiotic-resistant," as used herein, refers to
the ability of a microorganism to resist the toxic effects of an
antibiotic, usually due to a mutation. Resistance typically occurs
when the microorganism produces a protein that disables an
antibiotic or prevents transport of the antibiotic into the cell.
As used herein, the term also includes microorganisms that undergo
reversal of tolerance. An antibiotic-resistant microorganism is one
in which the minimum inhibitory concentration (MIC) is increased by
at least 10% relative to the average MIC of the non-resistant
strain.
[0055] The term "antibiotic-tolerant," as used herein, refers to
phenotypic variants of a microorganism produced stochastically in a
population, which are non-growing, doimant cells and therefore
tolerant of antibiotics.
[0056] The term "minimum inhibitory concentration (MIC)" refers to
the lowest concentration of a compound or molecule that prevents
visible growth of a bacterium.
[0057] The term "sequentially" refers to the administration of two
or more agents one after the other and close enough in time that
each of the agents exerts a biological activity on the other agent,
e.g., the two or more agents have an effect in combination.
[0058] One aspect of the invention relates to a composition
comprising a surfactant and an aminoglycoside antibiotic. The
composition may comprise more than one aminoglycoside antibiotic,
e.g., 1, 2, 3, 4, or 5 or more aminoglycoside antibiotics. The
composition may comprise more than one surfactant, e.g., 1, 2, 3,
4, or 5 or more surfactants. The composition may be a
pharmaceutical composition comprising a surfactant and an
aminoglycoside antibiotic together with a pharmaceutically
acceptable carrier.
[0059] In some embodiments, the surfactant is a membrane-modifying
surfactant, i.e., one that modifies the cell membrane to allow
increased penetration of an aminoglycoside antibiotic. In some
embodiments, the surfactant is a pore-forming biosurfactant (i.e.,
a naturally occurring molecule that is capable of forming a pore in
a cell membrane). In some embodiments, the pore-forming
biosurfactant may be a biosurfactant from a bacteria, e.g., a
Lactobacillus spp. or Streptococcus spp., e.g., L. easel, L.
lactis, L. fermentus RC 14, or S. thermophilus A. In some
embodiments, the pore-forming biosurfactant may be a rhamnolipid, a
xylolipid, or any combination thereof The rhamnolipid may be from
Pseudomonas aeruginosa. In some embodiments, the rhamnolipid may be
a mono-rhamnolipid, a di-rhamnolipid, or a combination thereof.
Rhamnolipids are a class of glycolipid that have a glycosyl head
group, in this case a rhamnose moiety, and a
3-(hydroxyalkanoyloxy)alkanoic acid (HAA) fatty acid tail, such as
3-hydroxydecanoic acid. There are two main classes of rhamnolipids:
mono-rhamnolipids and di-rhamnolipids, which consist of one or two
rhamnose groups respectively. In some embodiments, the rhamnolipids
are predominantly mono-rhamnolipids or di-rhamnolipids, e.g., at
least 70%, 75%, 80%, 85%, 90%, or more of mono-rhamnolipids or
di-rhamnolipids. In some embodiments, the rhamnolipids have 1 or 2
fatty acid tails that are predominantly C10 or C12, e.g., at least
70%, 75%, 80%, 85%, 90%, or more C10 or C12.
[0060] Surfactants (or surface-active substances) that may be
present are anionic, non-ionic, cationic and/or amphoteric
surfactants. Typical examples of anionic surfactants include, but
are not limited to, soaps, alkylbenzenesulfonates,
alkanesulfonates, olefin sulfonates, alkyl ether sulfonates,
glycerol ether sulfonates, a-methyl ester sulfonates, sulfo fatty
acids, alkyl sulphates, fatty alcohol ether sulphates, glycerol
ether sulphates, fatty acid ether sulphates, hydroxy mixed ether
sulphates, monoglyceride (ether) sulphates, fatty acid amide
(ether) sulphates, mono- and dialkyl sulfosuccinates, mono- and
dialkyl sulfosuccinamates, sulfotriglycerides, amide soaps, ether
carboxylic acids and salts thereof, fatty acid isethionates, fatty
acid sarcosinates, fatty acid taurides, N-acylamino acids, e.g.,
acyl lactylates, acyl tartrates, acyl glutamates and acyl
aspartates, alkyl oligoglucoside sulphates, protein fatty acid
condensates (in particular wheat-based vegetable products) and
alkyl (ether) phosphates. Examples of non-ionic surfactants
include, but are not limited to, fatty alcohol polyglycol ethers,
alkylphenol polyglycol ethers, fatty acid polyglycol esters, fatty
acid amide polyglycol ethers, fatty amine polyglycol ethers,
alkoxylated triglycerides, mixed ethers or mixed formals,
optionally partially oxidized alk(en)yl oligoglycosides or
glucoronic acid derivatives, fatty acid N-alkylglucamides, protein
hydrolysates (in particular wheat-based vegetable products), polyol
fatty acid esters, sugar esters, sorbitan esters, polysorbates and
amine oxides. Examples of amphoteric or zwitterionic surfactants
include, but are not limited to, alkylbetaines, alkylamidobetaines,
aminopropionates, aminoglycinates, imidazolinium-betaines and
sulfobetaines.
[0061] In some embodiments, the surfactant can be fatty alcohol
polyglycol ether sulphates, monoglyceride sulphates, mono- and/or
dialkyl sulfosuccinates, fatty acid isethionates, fatty acid
sarcosinates, fatty acid taurides, fatty acid glutamates,
alpha-olefinsulfonates, ether carboxylic acids, alkyl
oligoglucosides, fatty acid glucamides, alkylamidobetaines,
amphoacetals and/or protein fatty acid condensates.
[0062] Examples of zwitterionic surfactants include betaines, such
as N-alkyl-N,N-dimethylammonium glycinates, for example
cocoalkyldimethylammonium glycinate,
N-acylaminopropyl-N,N-dimethylammonium glycinates, for example
cocoacylaminopropyldimethylammonium glycinate, and
2-alkyl-3-carboxymethyl-3-hydroxyethylimidazolines having in each
case 8 to 18 carbon atoms in the alkyl or acyl group, and
cocoacylaminoethylhydroxyethyl-carboxymethyl glycinate.
[0063] In some embodiments, the surfactant can be a nonionogenic
surfactant selected from the following: the addition products of
from 2 to 30 mole of ethylene oxide and/or 0 to 5 mole of propylene
oxide onto linear fatty alcohols having 8 to 22 carbon atoms, onto
fatty acids having 12 to 22 carbon atoms, onto alkylphenols having
8 to 15 carbon atoms in the alkyl group, or onto alkylamines having
8 to 22 carbon atoms in the alkyl radical; alkyl and/or alkenyl
oligoglycosides having 8 to 22 carbon atoms in the alk(en)yl
radical and the ethoxylated analogs thereof; the addition products
of from 1 to 15 mole of ethylene oxide onto castor oil and/or
hydrogenated castor oil; the addition products of from 15 to 60
mole of ethylene oxide onto castor oil and/or hydrogenated castor
oil; partial esters of glycerol and/or sorbitan with unsaturated,
linear or saturated, branched fatty acids having 12 to 22 carbon
atoms and/or hydroxycarboxylic acids having 3 to 18 carbon atoms,
and the adducts thereof with 1 to 30 mole of ethylene oxide;
partial esters of polyglycerol (average degree of self-condensation
2 to 8), trimethylolpropane, pentaerythritol, sugar alcohols (e.g.,
sorbitol), alkyl glucosides (e.g., methyl glucoside, butyl
glucoside, lauryl glucoside), and polyglucosides (e.g., cellulose)
with saturated and/or unsaturated, linear or branched fatty acids
having 12 to 22 carbon atoms and/or hydroxycarboxylic acids having
3 to 18 carbon atoms, and the adducts thereof with 1 to 30 mole of
ethylene oxide; mixed esters of pentaerythritol, fatty acids,
citric acid and fatty alcohols and/or mixed esters of fatty acids
having 6 to 22 carbon atoms, methylglucose and polyols, preferably
glycerol or polyglycerol, mono-, di- and trialkyl phosphates, and
mono-, di- and/or tri-PEG alkyl phosphates and salts thereof; wool
wax alcohols; polysiloxane-polyalkyl-polyether copolymers and
corresponding derivatives; and block copolymers, e.g., polyethylene
glycol-30 dipolyhydroxystearates.
[0064] In some embodiments, the surfactant is a polyalkylene glycol
such as, for example, polyethylene glycol or polypropylene glycol.
In some embodiments, the surfactant is polyethylene glycol having a
molecular weight 100 Da to 5,000 Da, 200 Da to 2,500 Da, 300 Da to
1,000 Da, 400 Da to 750 Da, 550 Da to 650 Da, or about 600 Da.
[0065] In some embodiments, the surfactant is a poloxamer.
[0066] In some embodiments, the surfactant is composed of one or
more fatty alcohols. In some embodiments, the fatty alcohol is a
linear or branched C.sub.6 to C.sub.35 fatty alcohol. Examples of
fatty alcohols include, but are not limited to, capryl alcohol
(1-octanol), 2-ethyl hexanol, pelargonic alcohol (1-nonanol),
capric alcohol (1-decanol, decyl alcohol), undecyl alcohol
(1-undecanol, undecanol, hendecanol), lauryl alcohol (dodecanol,
1-dodecanol), tridecyl alcohol (1-tridecanol, tridecanol,
isotridecanol), myristyl alcohol (1-tetradecanol), pentadecyl
alcohol (1-pentadecanol, pentadecanol), cetyl alcohol
(1-hexadecanol), palmitoleyl alcohol (cis-9-hexadecen-1-ol),
heptadecyl alcohol (1-n-heptadecanol, heptadecanol), stearyl
alcohol (1-octadecanol), isostearyl alcohol
(16-methylheptadecan-1-ol), elaidyl alcohol (9E-octadecen-1-ol),
oleyl alcohol (cis-9-octadecen-1-ol), linoleyl alcohol
(9Z,12Z-octadecadien-1-ol), elaidolinoleyl alcohol
(9E,12E-octadecadien-1-ol), linolenyl alcohol
(9Z,12Z,15Z-octadecatrien-1-ol) elaidolinolenyl alcohol
(9E,12E,15-E-octadecatrien-1-ol), ricinoleyl alcohol
(12-hydroxy-9-octadecen-1-ol), nonadecyl alcohol (1-nonadecanol),
arachidyl alcohol (1-eicosanol), heneicosyl alcohol
(1-heneicosanol), behenyl alcohol (1-docosanol), erucyl alcohol
(cis-13-docosen-1-ol), lignoceryl alcohol (1-tetracosanol), ceryl
alcohol (1-hexacosanol), montanyl alcohol, cluytyl alcohol
(1-octacosanol), myricyl alcohol, melissyl alcohol
(1-triacontanol), geddyl alcohol (1-tetratriacontanol), or cetearyl
alcohol.
[0067] In some embodiments, the surfactant is a fatty acid, e.g.,
pahnitoleic acid or glycerol monolaureate.
[0068] The aminoglycoside antibiotic may be any aminoglycoside
antibiotic known to be therapeutically effective or which may be
therapeutically effective when combined with the surfactant. In
some embodiments, the aminoglycoside antibiotic is streptomycin,
kanamycin A, amikacin, tobramycin, dibekacin, gentamicin,
sisomicin, netilmicin, neomycin B, neomycin C, neomycin E
(paromomycin), or any combination thereof. In some embodiments, the
aminoglycoside antibiotic is tobramycin.
[0069] The composition may be a dosage form, e.g., a unit dosage
form. In some embodiments, the surfactant and aminoglycoside
antibiotic are both present in therapeutically effective amounts.
In some embodiments, the surfactant and aminoglycoside antibiotic
are both present in synergistic amounts, e.g., amounts that, when
administered to a subject, will produce a synergistic effect.
[0070] In certain embodiments, the surfactant and/or aminoglycoside
antibiotic is present in a sub-therapeutic amount (i.e., an amount
that does not provide antimicrobial activity) but produces a
therapeutic effect in combination. In some embodiments, the
surfactant and/or aminoglycoside antibiotic is present in an amount
that, by itself, is not therapeutic but renders a third antibiotic
therapeutically effective.
[0071] Another aspect of the invention relates to a kit comprising
a surfactant and an aminoglycoside antibiotic as discussed above.
The kit may comprise the surfactant and aminoglycoside antibiotic
in the same container or in separate containers.
[0072] Another aspect of the invention relates to a method of
increasing the sensitivity of a bacteria to an aminoglycoside
antibiotic, comprising contacting the bacteria with an effective
amount of a surfactant and the aminoglycoside antibiotic.
[0073] An additional aspect of the invention relates to a method of
treating a bacterial infection in a subject in need thereof,
comprising administering to the subject a therapeutically effective
amount of a surfactant and an aminoglycoside antibiotic.
[0074] Another aspect of the invention relates to a method of
reducing the risk of recurrence of a bacterial infection in a
subject in need thereof, comprising administering to the subject
having a bacterial infection a therapeutically effective amount of
a surfactant and an aminoglycoside antibiotic.
[0075] Another aspect of the invention relates to a method of
disrupting a biofilm, comprising contacting the biofilm with an
effective amount of a surfactant and an aminoglycoside
antibiotic.
[0076] In each of the methods of the invention, the surfactant and
the aminoglycoside antibiotic may be administered simultaneously,
e.g., in the same dosage form or in separate dosage forms. In each
of the methods of the invention, the surfactant and the
aminoglycoside antibiotic may be administered sequentially, but
close enough together in time to exert a biological effect on each
other. In each of the methods of the invention, the surfactant and
the aminoglycoside antibiotic may be administered in synergistic
amounts.
[0077] In each of the methods of the invention, the surfactant may
decrease the minimum inhibitory concentration of the aminoglycoside
antibiotic, e.g., by at least about 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, or 90%.
[0078] In some embodiments, the surfactant is administered before
the aminoglycoside antibiotic. In other embodiments, the
aminoglycoside antibiotic is administered before the surfactant.
The second molecule may be administered at any effective time point
after the first molecule is administered, e.g., about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60
minutes or about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours
after the first molecule is administered.
[0079] In certain embodiments of the methods of the invention, the
subject is one that has been diagnosed as being infected with
antibiotic-resistant or antibiotic-tolerant bacteria or is
suspected of being infected with antibiotic-resistant or
antibiotic-tolerant bacteria. The subject may not have been
previously treated for the infection. The subject may be
administered the surfactant first to prime the subject for
aminoglycoside antibiotic treatment.
[0080] In some embodiments, the subject has been treated with an
aminoglycoside antibiotic and the treatment has been ineffective
(e.g., the infection has not been reduced, has been reduced but not
eradicated, or was thought to have been eradicated but has
returned). The surfactant may be added to the treatment with the
same aminoglycoside antibiotic or with a different aminoglycoside
antibiotic.
[0081] In some embodiments, the subject may be immunocompromised or
otherwise have diminished ability to fight the infection.
[0082] The methods of the present invention may permit the
surfactant and/or the aminoglycoside antibiotic to be administered
at a dose that would not be therapeutically effective if
administered alone. The MIC of surfactant and/or aminoglycoside
antibiotic when provided together may be decreased by at least
about 5%, e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, or more relative to the MIC of either agent alone.
[0083] Another aspect of the invention relates to a composition
comprising a cell wall-hydrolyzing enzyme and a cell wall synthesis
inhibitor glycopeptide antibiotic. The composition may comprise
more than one cell wall synthesis inhibitor glycopeptide
antibiotic, e.g., 1, 2, 3, 4, or 5 or more cell wall synthesis
inhibitor glycopeptide antibiotics. The composition may comprise
more than one cell wall-hydrolyzing enzyme, e.g., 1, 2, 3, 4, or 5
or more cell wall-hydrolyzing enzymes.
[0084] In some embodiments, the cell wall-hydrolyzing enzyme may be
LasA protease, lysostaphin, an autolysin, or any combination
thereof In certain embodiments, the cell wall-hydrolyzing enzyme is
LasA protease.
[0085] In some embodiments, the cell wall synthesis inhibitor
glycopeptide antibiotic may be any cell wall synthesis inhibitor
glycopeptide antibiotic known to be therapeutically effective or
which may be therapeutically effective when combined with the cell
wall-hydrolyzing enzyme. In some embodiments, the aminoglycoside
antibiotic is may be vancomycin, teicoplanin, telavancin,
ramoplanin, decaplanin, oritavancin, dalbavancin, or any
combination thereof. In certain embodiments, the cell wall
synthesis inhibitor glycopeptide antibiotic is vancomycin.
[0086] The composition may be a dosage form, e.g., a unit dosage
form. In some embodiments, the cell wall-hydrolyzing enzyme and
cell wall synthesis inhibitor glycopeptide antibiotic are both
present in therapeutically effective amounts. In some embodiments,
the cell wall-hydrolyzing enzyme and cell wall synthesis inhibitor
glycopeptide antibiotic are both present in synergistic amounts,
e.g., amounts that, when administered to a subject, will produce a
synergistic effect.
[0087] In certain embodiments, the cell wall-hydrolyzing enzyme
and/or cell wall synthesis inhibitor glycopeptide antibiotic is
present in an amount that, by itself, is not therapeutic but
produces a therapeutic effect in combination. In some embodiments,
the cell wall-hydrolyzing enzyme and/or cell wall synthesis
inhibitor glycopeptide antibiotic is present in an amount that, by
itself, is not therapeutic but renders a third antibiotic
therapeutically effective.
[0088] Another aspect of the invention relates to a kit comprising,
a cell wall-hydrolyzing enzyme and a cell wall synthesis inhibitor
glycopeptide antibiotic as discussed above. The kit may comprise
the cell wall-hydrolyzing enzyme and cell wall synthesis inhibitor
glycopeptide antibiotic in the same container or in separate
containers.
[0089] Another aspect of the invention relates to a method of
increasing the sensitivity of a bacteria to a cell wall synthesis
inhibitor glycopeptide antibiotic, comprising contacting the
bacteria with an effective amount of a cell wall-hydrolyzing enzyme
and the cell wall synthesis inhibitor glycopeptide antibiotic.
[0090] An additional aspect of the invention relates to a method of
treating a bacterial infection in a subject in need thereof,
comprising administering to the subject a therapeutically effective
amount of a cell wall-hydrolyzing enzyme and a cell wall synthesis
inhibitor glycopeptide antibiotic.
[0091] Another aspect of the invention relates to a method of
reducing the risk of recurrence of a bacterial infection in a
subject in need thereof, comprising administering to the subject
having a bacterial infection a therapeutically effective amount of
a cell wall-hydrolyzing enzyme and a cell wall synthesis inhibitor
glycopeptide antibiotic.
[0092] Another aspect of the invention relates to a method of
disrupting a biofilm, comprising contacting the biofilm with an
effective amount of a cell wall-hydrolyzing enzyme and a cell wall
synthesis inhibitor glycopeptide antibiotic.
[0093] In each of the methods of the invention, the cell
wall-hydrolyzing enzyme and the cell wall synthesis inhibitor
glycopeptide antibiotic may be administered simultaneously, e.g.,
in the same dosage form or in separate dosage forms. In each of the
methods of the invention, the cell wall-hydrolyzing enzyme and the
cell wall synthesis inhibitor glycopeptide antibiotic may be
administered sequentially, but close enough together in time to
exert a biological effect on each other. In each of the methods of
the invention, the cell wall-hydrolyzing enzyme and the cell wall
synthesis inhibitor glycopeptide antibiotic may be administered in
synergistic amounts.
[0094] In each of the methods of the invention, the cell
wall-hydrolyzing enzyme may decrease the minimum inhibitory
concentration of the cell wall synthesis inhibitor glycopeptide
antibiotic.
[0095] In some embodiments, the cell wall-hydrolyzing enzyme is
administered before the cell wall synthesis inhibitor glycopeptide
antibiotic. In other embodiments, the cell wall synthesis inhibitor
glycopeptide antibiotic is administered before the cell
wall-hydrolyzing enzyme. The second molecule may be administered at
any effective time point after the first molecule is administered,
e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,
45, 50, 55, or 60 minutes or about 1,5, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, or 12 hours after the first molecule is administered.
[0096] In certain embodiments of the methods of the invention, the
subject is one that has been diagnosed as being infected with
antibiotic-resistant or antibiotic-tolerant bacteria or is
suspected of being infected with antibiotic-resistant or
antibiotic-tolerant bacteria. The subject may not have been
previously treated for the infection. The subject may be
administered the cell wall-hydrolyzing enzyme first to prime the
subject for cell wall synthesis inhibitor glycopeptide antibiotic
treatment.
[0097] In some embodiments, the subject has been treated with a
cell wall synthesis inhibitor glycopeptide antibiotic and the
treatment has been ineffective (e.g., the infection has not been
reduced, has been reduced but not eradicated, or was thought to
have been eradicated but has returned). The cell wall-hydrolyzing
enzyme may be added to the treatment with the same cell wall
synthesis inhibitor glycopeptide antibiotic or with a different
cell wall synthesis inhibitor glycopeptide antibiotic.
[0098] In some embodiments, the subject may be immunocompromised or
otherwise have diminished ability to fight the infection.
[0099] The methods of the present invention may permit the cell
wall-hydrolyzing enzyme and/or the cell wall synthesis inhibitor
glycopeptide antibiotic to be administered at a dose that that
would not be therapeutically effective if administered alone. The
MIC of the cell wall-hydrolyzing enzyme and/or cell wall synthesis
inhibitor glycopeptide antibiotic when provided together may be
decreased by at least about 5%, e.g., at least about 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, or more relative to the MIC of either
agent alone.
[0100] The microorganism, e.g., bacteria, treated by the present
invention may be any microorganism known in the art. In some
embodiments, the microorganism is an antibiotic-resistant or
antibiotic-tolerant strain. In certain embodiments, the bacteria is
a gram-negative bacteria. In certain embodiments, the bacteria is a
gram-positive bacteria, e.g., Staphylococcus aureus, e.g.,
methicillin-resistant Staphylococcus aureus. Pathogenic bacteria
and other microorganisms include, but are not limited to,
Rickettsia, Chlamydia, Mycobacteria, Clostridia, Corynebacteria,
Mycoplasma, Ureaplasma, Legionella, Shigella, Salmonella,
pathogenic Escherichia coli species, Bordatella, Neisseria,
Treponema, Bacillus, Haemophilus, Moraxella, Vibrio, Staphylococcus
spp., Streptococcus spp., Campylobacter spp., Borrelia spp.,
Leptospira spp., Erlichia spp., Klebsiella spp., Pseudomonas spp.,
Helicobacter spp., and any other pathogenic microorganism now known
or later identified (see, e.g., Microbiology, Davis et al, Eds.,
4.sup.th ed., Lippincott, N.Y., 1990, the entire contents of which
are incorporated herein by reference for the teachings of
pathogenic microorganisms). Specific examples of microorganisms
include, but are not limited to, Helicobacter pylori, Chlamydia
pneumoniae, Chlamydia trachomatis, Ureaplasma urealvticum,
Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus
pyogenes, Streptococcus pneumoniae, Streptococcus viridans,
Enterococcus faecalis, Neisseria meningitidis, Neisseria
gonorrhoeae, Treponema pallidum, Bacillus anthracis, Salmonella
Vibrio cholera, Pasteurella pestis (Yersinia pestis), Pseudomonas
aeruginosa, Campylobacter jejuni, Clostridium difficile,
Clostridium botulinum, Mycobacterium tuberculosis, Borrelia
burgdorferi, Haemophilus ducreyi, Corynebacterium diphtheria,
Bordetella pertussis, Bordetella parapertussis, Bordetella
bronchiseptica, Haemophilus influenza, and enterotoxic Escherichia
coll. In some embodiments, the bacteria is Staphylococcus aureus.
In some embodiments, the bacteria is methicillin-resistant
Staphylococcus aureus. In certain embodiments, the bacteria may be
gram-negative bacteria or gram-positive bacteria.
[0101] In certain embodiments, the surfactant and aminoglycoside
antibiotic or the cell wall-hydrolyzing enzyme and cell wall
synthesis inhibitor glycopeptide antibiotic of the invention are
administered directly to a subject. In some embodiments, the
compounds will be suspended in a pharmaceutically-acceptable
carrier (e.g., physiological saline) and administered orally or by
intravenous infusion, or administered subcutaneously,
intramuscularly, intrathecally, intraperitoneally, intrarectally,
intravaginally, intranasally, intragastrically, intratracheally, or
intrapulmonarily. In another embodiment, the intratracheal or
intrapulmonary delivery can be accomplished using a standard
nebulizer, jet nebulizer, wire mesh nebulizer, dry powder inhaler,
or metered dose inhaler. The agents can be delivered directly to
the site of the disease or disorder, such as lungs, kidney, or
intestines. The dosage required depends on the choice of the route
of administration; the nature of the formulation; the nature of the
patient's illness; the subject's size, weight, surface area, age,
and sex; other drugs being administered; and the judgment of the
attending physician. Suitable dosages for each agent are in the
range of 0.01-100.0 .mu.g/kg. Wide variations in the needed dosage
are to be expected in view of the variety of antibiotics available
and the differing efficiencies of various routes of administration.
For example, oral administration would be expected to require
higher dosages than administration by i.v. injection. Variations in
these dosage levels can be adjusted using standard empirical
routines for optimization as is well understood in the art.
Administrations can be single or multiple (e.g., 2-, 3-, 4-, 6-,
8-, 10-; 20-, 50-, 100-, 150-, or more fold). Encapsulation of the
surfactant and aminoglycoside antibiotic or the cell
wall-hydrolyzing enzyme and cell wall synthesis inhibitor
glycopeptide antibiotic in a suitable delivery vehicle (e.g.,
polymeric microparticles or implantable devices) may increase the
efficiency of delivery, particularly for oral delivery.
[0102] As a further aspect, the invention provides pharmaceutical
formulations and methods of administering the same to achieve any
of the therapeutic effects (e.g., treatment of infection) discussed
above. The pharmaceutical formulation may comprise any of the
reagents discussed above in a pharmaceutically acceptable
carrier.
[0103] By "pharmaceutically acceptable" it is meant a material that
is not biologically or otherwise undesirable, i.e., the material
can be administered to a subject without causing any undesirable
biological effects such as toxicity.
[0104] The formulations of the invention can optionally comprise
medicinal agents, pharmaceutical agents, carriers, adjuvants,
dispersing agents, diluents, and the like.
[0105] The compounds of the invention can be formulated for
administration in a pharmaceutical carrier in accordance with known
techniques. See, e.g., Remington, The Science And Practice of
Pharmacy (21.sup.st Ed. 2006). In the manufacture of a
pharmaceutical formulation according to the invention, the compound
(including the physiologically acceptable salts thereof) is
typically admixed with, inter alia, an acceptable carrier. The
carrier can be a solid or a liquid, or both, and is preferably
formulated with the compound as a unit-dose foimulation, for
example, a tablet, which can contain from 0.01 or 0.5% to 95% or
99% by weight of the compound. One or more compounds can be
incorporated in the formulations of the invention, which can be
prepared by any of the well-known techniques of pharmacy.
[0106] The formulations of the invention include those suitable for
oral, rectal, topical, buccal (e.g., sub-lingual), vaginal,
parenteral (e.g., subcutaneous, intramuscular including skeletal
muscle, cardiac muscle, diaphragm muscle and smooth muscle,
intradermal, intravenous, intraperitoneal), topical (i.e., both
skin and mucosal surfaces, including airway surfaces), intranasal,
transdermal, intraarticular, intrathecal, and inhalation
administration, administration to the liver by intraportal
delivery, as well as direct organ injection (e.g., into the liver,
into the brain for delivery to the central nervous system, or into
the pancreas) or injection into a body cavity. The most suitable
route in any given case will depend on the nature and severity of
the condition being treated and on the nature of the particular
compound which is being used.
[0107] For injection, the carrier will typically be a liquid, such
as sterile pyrogen-free water, pyrogen-free phosphate-buffered
saline solution, bacteriostatic water, or Cremophor EL[R] (BASF,
Parsippany, N.J.). For other methods of administration, the carrier
can be either solid or liquid.
[0108] For oral administration, the compound can be administered in
solid dosage forms, such as capsules, tablets, and powders, or in
liquid dosage forms, such as elixirs, syrups, and suspensions.
Compounds can be encapsulated in gelatin capsules together with
inactive ingredients and powdered carriers, such as glucose,
lactose, sucrose, mannitol, starch, cellulose or cellulose
derivatives, magnesium stearate, stearic acid, sodium saccharin,
talcum, magnesium carbonate and the like. Examples of additional
inactive ingredients that can be added to provide desirable color,
taste, stability, buffering capacity, dispersion or other known
desirable features are red iron oxide, silica gel, sodium lauryl
sulfate, titanium dioxide, edible white ink and the like. Similar
diluents can be used to make compressed tablets. Both tablets and
capsules can be manufactured as sustained release products to
provide for continuous release of medication over a period of
hours. Compressed tablets can be sugar coated or film coated to
mask any unpleasant taste and protect the tablet from the
atmosphere, or enteric- coated for selective disintegration in the
gastrointestinal tract. Liquid dosage forms for oral administration
can contain coloring and flavoring to increase patient
acceptance.
[0109] Formulations suitable for buccal (sub-lingual)
administration include lozenges comprising the compound in a
flavored base, usually sucrose and acacia or tragacanth; and
pastilles comprising the compound in an inert base such as gelatin
and glycerin or sucrose and acacia.
[0110] Formulations of the present invention suitable for
parenteral administration comprise sterile aqueous and non-aqueous
injection solutions of the compound, which preparations are
preferably isotonic with the blood of the intended recipient. These
preparations can contain anti-oxidants, buffers, bacteriostats and
solutes which render the formulation isotonic with the blood of the
intended recipient. Aqueous and non-aqueous sterile suspensions can
include suspending agents and thickening agents. The formulations
can be presented in unit dose or multi-dose containers, for example
sealed ampoules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, saline or water-for-injection
immediately prior to use.
[0111] Extemporaneous injection solutions and suspensions can be
prepared from sterile powders, granules and tablets of the kind
previously described. For example, in one aspect of the present
invention, there is provided an injectable, stable, sterile
composition comprising a compound of the invention, in a unit
dosage form in a sealed container. The compound or salt is provided
in the form of a lyophilizate which is capable of being
reconstituted with a suitable pharmaceutically acceptable carrier
to form a liquid composition suitable for injection thereof into a
subject. The unit dosage form typically comprises from about 1 mg
to about 10 grams of the compound or salt. When the compound or
salt is substantially water-insoluble, a sufficient amount of
emulsifying agent which is pharmaceutically acceptable can be
employed in sufficient quantity to emulsify the compound or salt in
an aqueous carrier. One such useful emulsifying agent is
phosphatidyl choline.
[0112] Formulations suitable for rectal administration are
preferably presented as unit dose suppositories. These can be
prepared by admixing the compound with one or more conventional
solid carriers, for example, cocoa butter, and then shaping the
resulting mixture.
[0113] Formulations suitable for topical application to the skin
preferably take the form of an ointment, cream, lotion, paste, gel,
spray, aerosol, or oil. Carriers which can be used include
petroleum jelly, lanoline, polyethylene glycols, alcohols,
transdermal enhancers, and combinations of two or more thereof.
[0114] Formulations suitable for transdermal administration can be
presented as discrete patches adapted to remain in intimate contact
with the epidermis of the recipient for a prolonged period of time.
Formulations suitable for transdermal administration can also be
delivered by iontophoresis (see, for example, Tyle, Pharm. Res.
3:318 (1986)) and typically take the form of an optionally buffered
aqueous solution of the compounds. Suitable formulations comprise
citrate or bis/tris buffer (pH 6) or ethanol/water and contain from
0.1 to 0.2M of the compound.
[0115] The compound can alternatively be formulated for nasal
administration or otherwise administered to the lungs of a subject
by any suitable means, e.g., administered by an aerosol suspension
of respirable particles comprising the compound, which the subject
inhales. The respirable particles can be liquid or solid. The term
"aerosol" includes any gas-borne suspended phase, which is capable
of being inhaled into the bronchioles or nasal passages.
Specifically, aerosol includes a gas-borne suspension of droplets,
as can be produced in a metered dose inhaler or nebulizer, or in a
mist sprayer. Aerosol also includes a thy powder composition
suspended in air or other carrier gas, which can be delivered by
insufflation from an inhaler device, for example. See Ganderton
& Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood
(1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier
Systems 6:273-313; and Raeburn et al., Pharmacol. Toxicol. Meth.
27:143 (1992). Aerosols of liquid particles comprising the compound
can be produced by any suitable means, such as with a
pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is
known to those of skill in the art. See, e.g., U.S. Pat. No.
4,501,729. Aerosols of solid particles comprising the compound can
likewise be produced with any solid particulate medicament aerosol
generator, by techniques known in the pharmaceutical art.
[0116] Alternatively, one can administer the compound in a local
rather than systemic manner, for example, in a depot or
sustained-release formulation.
[0117] Further, the present invention provides liposomal
formulations of the compounds disclosed herein and salts thereof.
The technology for forming liposomal suspensions is well known in
the art. When the compound or salt thereof is an aqueous-soluble
salt, using conventional liposome technology, the same can be
incorporated into lipid vesicles. In such an instance, due to the
water solubility of the compound or salt, the compound or salt will
be substantially entrained within the hydrophilic center or core of
the liposomes. The lipid layer employed can be of any conventional
composition and can either contain cholesterol or can be
cholesterol-free. When the compound or salt of interest is
water-insoluble, again employing conventional liposome formation
technology, the salt can be substantially entrained within the
hydrophobic lipid bilayer which forms the structure of the
liposome. In either instance, the liposomes which are produced can
be reduced in size, as through the use of standard sonication and
homogenization techniques.
[0118] The liposomal formulations containing the compounds
disclosed herein or salts thereof, can be lyophilized to produce a
lyophilizate which can be reconstituted with a pharmaceutically
acceptable carrier, such as water, to regenerate a liposomal
suspension.
[0119] In the case of water-insoluble compounds, a pharmaceutical
composition can be prepared containing the water-insoluble
compound, such as for example, in an aqueous base emulsion. In such
an instance, the composition will contain a sufficient amount of
pharmaceutically acceptable emulsifying agent to emulsify the
desired amount of the compound. Particularly useful emulsifying
agents include phosphatidyl cholines and lecithin.
[0120] In particular embodiments, the compound is administered to
the subject in a therapeutically effective amount, as that term is
defined above. Dosages of pharmaceutically active compounds can be
determined by methods known in the art, see, e.g., Remington's
Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa). The
therapeutically effective dosage of any specific compound will vary
somewhat from compound to compound, and patient to patient, and
will depend upon the condition of the patient and the route of
delivery. As a general proposition, a dosage from about 0.1 to
about 50 mg/kg will have therapeutic efficacy, with all weights
being calculated based upon the weight of the compound, including
the cases where a salt is employed. Toxicity concerns at the higher
level can restrict intravenous dosages to a lower level such as up
to about 10 mg/kg, with all weights being calculated based upon the
weight of the compound, including the cases where a salt is
employed. A dosage from about 10 mg/kg to about 50 mg/kg can be
employed for oral administration. Typically, a dosage from about
0.5 mg/kg to 5 mg/kg can be employed for intramuscular injection.
Particular dosages are about 1 .mu.mol/kg to 50 .mu.mol/kg, and
more particularly to about 22 .mu.mol/kg and to 33 .mu.mol/kg of
the compound for intravenous or oral administration,
respectively.
[0121] In particular embodiments of the invention, more than one
administration (e.g., two, three, four, or more administrations)
can be employed over a variety of time intervals (e.g., hourly,
daily, weekly, monthly, etc.) to achieve therapeutic effects.
[0122] The present invention finds use in veterinary and medical
applications. Suitable subjects include both avians and mammals,
with mammals being preferred. The term "avian" as used herein
includes, but is not limited to, chickens, ducks, geese, quail,
turkeys, and pheasants. The term "mammal" as used herein includes,
but is not limited to, humans, bovines, ovines, caprines, equines,
felines, canines, lagomorphs, etc. Human subjects include neonates,
infants, juveniles, and adults.
[0123] The present invention is more particularly described in the
following examples that are intended as illustrative only since
numerous modifications and variations therein will be apparent to
those skilled in the art.
EXAMPLE 1
Interspecies Interaction Determines S. aureus Antibiotic
Susceptibility
[0124] Staphylococcus aureus is a major human pathogen responsible
for numerous chronic and relapsing infections. These infections
often fail to respond to antibiotic treatment, even in the apparent
absence of antibiotic resistance. S. aureus frequently co-exists
with the opportunistic pathogen Pseudomonas aeruginosa in burns,
chronic wounds and the cystic fibrosis lung. Here, it is
demonstrated that interaction with P. aeruginosa alters antibiotic
susceptibility of S. aureus through a number of distinct pathways.
At physiologically relevant concentrations, P. aeruginosa secondary
metabolite 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) induces
tolerance of S. aureus to multiple antibiotics classes through
respiratory inhibition and reduction of cellular ATP levels.
Conversely, the P. aeruginosa bacteriolytic enzyme LasA potentiates
killing and lysis of S. aureus by vancomycin. Furthermore. P.
aeruginosa rhamnolipids facilitate the proton-motive
force-independent uptake of tobramycin, promoting eradication of S.
aureus persister populations. It was found here that the overall
ability of P. aeruginosa to alter S. aureus antibiotic
susceptibility is dependent on the production of HQNO, LasA and
rhamnolipids, all of which are highly variable among clinical
isolates examined. These findings demonstrate that antibiotic
susceptibility is dependent not only on the genotype of the
pathogen being targeted, but also on that of co-infecting
microorganisms in the infection environment.
[0125] Accurate prediction of antimicrobial efficacy is essential
for successful treatment of bacterial infection. Here it is shown
that a single interspecies interaction between S. aureus and P.
aeruginosa can completely transform the antibiotic susceptibility
profile of S. aureus . Through multiple distinct mechanisms, P.
aeruginosa can antagonize or potentiate the efficacy of multiple
classes of antibiotics against S. aureus . Further, it is shown
that the capacity of P. aeruginosa to alter antibiotic
susceptibility of S. aureus is highly variable in clinical
isolates. This work suggests that the efficacy of antibiotic
treatment in polymicrobial infection is determined on the community
level with interspecies interactions playing an important and as
yet unappreciated role.
Materials and Methods
[0126] Bacterial strains and growth conditions. S. aureus strain
HG003 was cultured aerobically in Mueller-Hinton broth (MHB) at
37.degree. C. with shaking at 225 rpm. For anaerobic growth,
overnight cultures were washed twice with PBS and diluted into 5 ml
of pre-warmed (37.degree. C.) TSB+100 mM MOPS (pH 7) to an
OD.sub.600 of 0.05. Cultures were prepared in triplicate in
16.times.150 mm glass tubes containing 1 mm stir bars. Following
dilution, cultures were immediately transferred into a Coy
anaerobic chamber and grown at 37.degree. C. with stirring. P.
aeruginosa strains were grown aerobically in MHB at 37.degree. C.
with shaking at 225 rpm. Burn wound isolates represent the first
positive Pseudomonal wound cultures obtained from 5 unique patients
admitted to the NC Jaycee Burn Center with a total body surface
area burn .gtoreq.20% and/or inhalational injury after obtaining
informed consent. Cystic fibrosis isolates were collected from 5
patients at the UNC medical center. Isolates were cultured from
sputum or bronchoalveolar lavage (BAL) from patients with cystic
fibrosis after obtaining informed consent.
[0127] Antibiotic survival assays. To prepare sterile supernatants,
S. aureus and P. aeruginosa strains were grown in MHB at 37.degree.
C. with shaking at 225 rpm for .about.20h. The cultures were
pelleted and supernatants were passed through a 0.2 .mu.m filter.
HG003 was grown to .about.5.times.10.sup.7 (for cell wall acting
antibiotics) or .about.2.times.10.sup.8 cfu/ml (for all other
antibiotics) in 3 ml MHB under aerobic conditions or in 5 ml
TSB+100 mM MOPS under anaerobic conditions. Cells were pre-treated
with 0.5 ml sterile supernatant (or 0.83 ml for anaerobic cultures)
and returned to the incubator for a further 30 min. An aliquot was
plated to enumerate cfu before the addition of antibiotics.
Antibiotics were added at concentrations similar to the Cmax in
humans at recommended dosing; ciprofloxacin 2.34 .mu.g/ml,
tobramycin 58 .mu.g/ml, oxacillin 50 .mu.g/ml, vancomycin 50
.mu.g/ml. Ciprofloxacin concentration was increased to 4.68
.mu.g/ml when cells were grown in TSB+100 mM MOPS to account for
any decrease in pH where ciprofloxacin killing activity is reduced.
At indicated times, an aliquot was removed and washed with 1% NaCl.
Cells were serially diluted and plated to enumerate survivors.
Where indicated sterile supernatant was heat-inactivated at
95.degree. C. for 10 min before addition to culture. Where
indicated, pyocyanin 100 HQNO 11.5 .mu.M, sodium cyanide 150 .mu.M
or rhamnolipids 10-50 .mu.g/ml (50/50 mix of mono- and
di-rhamnolipids, Sigma) were added in place of supernatant.
Concentrations of respiratory toxins represent levels detected in
the sputum of cystic fibrosis patients.
[0128] Promoter induction measurement. S. aureus strain HG003
harboring gfp promoter plasmid PpflB::gfp were grown to
.about.2.times.10.sup.8 cfu/ml in 3 ml MHB containing
chloramphenicol 10 g/ml. Cultures were treated with 0.5 ml
supernatant from HG003, PAO1, PA14 or P. aeruginosa clinical
isolates as indicated. 200 .mu.l culture was added to the wells of
a clear bottom, black side 96-well plate. The plate was placed in a
Biotek Synergy HI microplate reader at 37.degree. C. with shaking.
Absorbance (OD.sub.600) and GFP fluorescence (emission 528 nm and
excitation 485 nm) were measured every 1 h for 16 h. GFP values
were divided by OD.sub.600.
[0129] ATP assays. HG003 was grown to .about.2.times.10.sup.8
cfu/ml in 3 ml MHB and pre-treated with 0.5 ml sterile supernatant
from S. aureus HG003 or P. aeruginosa PAO1 or PA14. ATP levels of
the cultures were measured after 1.5 h as described previously
using a Promega BacTiter Glo kit according to the manufacturer's
instructions. P-values are indicated.
[0130] Vancomvcin lysis assay. HG003 was grown to
.about.2.times.10.sup.8 cfu/ml in 3 ml MHB and pre-treated with 0.5
ml sterile supernatant from S. aureus HG003, P. aeruginosa PAO1,
PA14 or P. aeruginosa clinical isolates as indicated. Cells were
incubated for a further 30 min before addition of vancomycin 50
.mu.g/ml. 200 .mu.l aliquots were added to the wells of a clear
96-well plate and placed in a Biotek Synergy H1 microplate reader.
Absorbance (OD.sub.600) was measured every 1 h for 16 h.
[0131] Tobramycin-Texas Red Uptake. Tobramycin-Texas Red was made
as described previously. S. aureus strain HG003 was grown to
mid-exponential phase and then incubated with or without 30
.mu.g/ml rhanmolipids for 30 min. Cells were plated to enumerate
cfu prior to addition of Texas-Red tobramycin at a final
concentration of 58 .mu.g/ml. After 1 h, an aliquot of cells was
removed, washed twice in 1% NaCl and plated to enumerate survivors.
The remaining aliquot was analyzed for Texas Red uptake on a BD
Fortessa flow cytometer. 30,000 events were recorded. Figures were
generated using FSC Express 6 Flow.
[0132] Western blot analysis of LasA. P. aeruginosa strains were
grown in MHB media for .about.20 h. Cultures were normalized to
OD.sub.600 2.0, pelleted and supernatants were passed through a 0.2
.mu.m filter. Supernatants were boiled in SDS-sample buffer and run
on a 4-12% bis-tris acrylamide gel (Invitrogen). Protein was
transferred onto a PVDF membrane and LasA was detected using rabbit
polyclonal anti-LasA antibodies (LifeSpan BioSciences, Inc.).
[0133] Staphvlolytic assay. Staphylolytic assay was modified from
Grande et at. Stationary phase S. aureus strain HG003 was heat
killed at 95.degree. C. for 20 min. Cells were pelleted and
resuspended in 20 mM Tris-HCl (pH 8.0) at an OD.sub.595 0.8-1. P.
aeruginosa strains were cultured in MHB media for .about.20 h.
Cultures were normalized to OD.sub.600 2.0, pelleted and
supernatants were passed through a 0.2 .mu.m filter. 17 .mu.l
sterile supernatant was added to 100 .mu.l heat-killed cells.
OD.sub.595 was measured at time 0 and after 2 h and % cell lysis
was determined. The values shown represent the average of
biological triplicates.
[0134] Rhamnolipid Quantification. P. aeruginosa rhamnolipid
production was quantified utilizing a drop collapse assay, as
previously described. Briefly, clarified supernatants from
overnight cultures of P. aeruginosa strains were serially diluted
(1:1) with de-ionized water plus 0.005% crystal violet for
visualization. 25 .mu.l aliquots of each dilution were spotted on
to the underside of a Petri dish plate and tilted to a 90.degree.
angle. Surfactant scores represent the reciprocal of the highest
dilution at which a collapsed drop migrated down the surface of the
plate.
[0135] Minimum inhibitory concentration (MIC) assays. MICs were
determined using the microdilution method. Briefly,
.about.5.times.10.sup.5 cfu were incubated with varying
concentrations of ciprofloxacin, tobramycin, oxacillin or
vancomycin in a total volume of 200 pl MHB in a 96-well plate.
Where indicated, 34 .mu.l MHB was replaced with sterile P.
aeruginosa or S. aureus supernatant or purified rhamnolipids at a
final concentration of 10-50 .mu.g/ml. MICs were determined
following incubation at 37.degree. C. for 24 h.
Results
[0136] P. aeruginosa Supernatant Alters S. aureus Susceptibility to
Antibiotic Killing
[0137] Co-infections of P. aeruginosa and S. aureus within chronic
wounds and the CF lung are generally more virulent and/or more
difficult to treat than infections caused by either pathogen alone.
The potential impact of this interspecies interaction on antibiotic
susceptibility of S. aureus was analyzed. Cultures of S. aureus
HG003 were grown to exponential phase and treated with sterile
supernatants from P. aeruginosa PAO1, P. aeruginosa PA14 or S.
aureus HG003 (control) overnight cultures for 30 minutes. Cultures
were then challenged with various bactericidal antibiotics at
physiologically relevant concentrations. It was found that P.
aeruginosa PAO1 or PA14 supernatant induced full tolerance of the
population to ciprofloxacin--a fluoroquinolone and oxacillin--a
.beta.-lactam. This is in stark contrast to S. aureus supernatant
treated control cultures, wherein at least 99% of the cells were
killed by the antibiotic (FIGS. 1A and 1B). Interestingly, P.
aeruginosa supernatant did not protect S. aureus from vancomycin
killing. On the contrary, the presence of PA14 or PA01 supernatant
during vancomycin challenge resulted in a 2- and 4-log increase in
cell death, respectively, relative to the S. aureus supernatant
control (FIG. 1C). Minimum inhibitory concentration (MIC) assays
were performed in the presence and absence of P. aeruginosa
supernatant, and the MIC of oxacillin, vancomycin and ciprofloxacin
were not affected by the presence of P. aeruginosa , demonstrating
the phenotype is associated with tolerance but not resistance
(Table 1).
[0138] The presence of P. aeruginosa has been associated with
increased aminoglycoside resistance in S. aureus . MIC assays
confirmed that P. aeruginosa supernatant induces an 8-fold increase
in tobramycin resistance in S. aureus . Paradoxically, P.
aeruginosa supernatant did not confer protection against tobramycin
killing (Table 1, FIG. 1D). Together, these data highlight the
complex influence P. aeruginosa exerts on antibiotic susceptibility
of S. aureus.
P. aeruginosa Induces Multidrug Tolerance in S. aureus Through
Respiratory Inhibition
[0139] The bactericidal activity of many major classes of
antibiotics is dependent on the activity of ATP-dependent processes
including cell wall biosynthesis, DNA replication, transcription
and translation. Low intracellular ATP concentrations lead to
decreased activity of these pathways resulting in less
antibiotic-induced damage and increased antibiotic tolerance. It
was previously shown that the presence of P. aeruginosa pushes S.
aureus towards fermentative mode of growth, even under aerobic
conditions. To test this, the fermentation-specific promoter for
pyruvate acetyltransferase (pflB) was cloned from S. aureus
upstream ofgfp in a low-copy plasmid. It was found that pflB
expression is induced in S. aureus in response to P. aeruginosa
supernatant (FIG. 2A). Fermentation is a far less efficient pathway
for ATP production, yielding 2 ATP molecules for every glucose
molecule metabolized, compared to the 32 ATP per glucose generated
through aerobic respiration. Direct intracellular ATP
quantification of cultures treated with P. aeruginosa or S. aureus
supernatant revealed that P. aeruginosa supernatant induces
significant depletion of S. aureus intracellular ATP (FIG. 2B).
[0140] P. aeruginosa secondary metabolites pyocyanin, HCN and HQNO
inhibit the S. aureus electron transport chain, thereby inhibiting
aerobic respiration. Furthermore, culture of S. aureus in the
presence of purified HQNO results in increased resistance to
tobramycin. It was reasoned that these respiratory toxins may be
responsible for inducing a low ATP, antibiotic tolerant state in S.
aureus during exposure to P. aeruginosa supernatant. To investigate
this possibility, we created mutants in genes pqsL, phzS and hcnC
in PA14. These mutants are incapable of producing HQNO, pyocyanin
and hydrogen cyanide, respectively. It was found that mutation of
pqsL (HQNO negative) drastically reduced the capacity of the
supernatant to induce S. aureus antibiotic tolerance suggesting
conditions tolerance to ciprofloxacin is primarily mediated by HQNO
(FIG. 2C). Double (FIG. 7A) and triple mutants (FIG. 2C) deficient
in the biosynthesis of HQNO, pyocyanin and hydrogen cyanide were
constructed. Although deletion of pqsL has the most dramatic effect
on the ability of P. aeruginosa to induce tolerance in S. aureus ,
a .DELTA.pqsLphzShenC triple mutant was further reduced in its
ability to confer protection to antibiotic killing (FIG. 2C). The
ability of P. aeruginosa supernatant to induce antibiotic tolerance
in S. aureus under anaerobic conditions was examined. As expected,
no protection from antibiotic killing was observed following
pre-treatment with PA14 supernatant during anoxic growth in the
absence of a terminal electron acceptor (FIG. 7B), supporting the
conclusion that P. aeruginosa -mediated induction of S. aureus
antibiotic tolerance is due to the inhibition of S. aureus
respiration.
[0141] Next, exponential phase S. aureus cultures were exposed to
concentrations of HQNO, pyocyanin or cyanide previously detected in
the sputum of cystic fibrosis patients with active P. aeruginosa
infection, then challenged these cultures with antibiotics. It was
found that all three compounds were capable of inducing tolerance
of S. aureus to ciprofloxacin (FIG. 2D). Similar levels of
tolerance were observed for oxacillin, tobramycin and vancomycin,
with HQNO inducing the most robust tolerance to antibiotic killing
(FIGS. 7C-7E).
P. aeruginosa LasA Endopeptidase Potentiates Vancomycin
Bactericidal Activity Against S. aureus
[0142] The presence of purified HQNO, pyocyanin or NaCN confers
protection to S. aureus against all antibiotics tested, including
vancomycin. However, it was observed that P. aeruginosa supernatant
significantly potentiates vancomycin killing of S. aureus (FIG.
1C). It was reasoned that P. aeruginosa supernatant must contain
one or more additional factors that dominate the protective
influence of P. aeruginosa respiratory toxins. Heat-inactivated
PAO1 supernatant failed to potentiate vancomycin killing of S.
aureus cultures, implicating an extracellular protein in the
phenotype (FIG. 3A). The potentiation of vancomycin killing was
accompanied by total lysis of the culture over time (FIG. 3B).
Importantly, no lysis was observed for cells treated with P.
aeruginosa supernatant in the absence of vancomycin (FIG. 3B). Due
to the lytic nature of the killing, LasA, an endopeptidase produced
by P. aeruginosa , which was previously shown to attack the cell
wall of S. aureus during in vivo competition, became of interest.
The capacity of supernatant from a lasA mutant in PAO1 to synergize
with vancomycin was examined. Interestingly, it was found that no
significant killing by vancomycin occurred under these conditions
compared to a 3-log reduction in S. aureus cfu in the presence of
the PAO1 wild-type supernatant (FIGS. 3A and 3C). It appears that
the combination of vancomycin inhibition of peptidoglycan
cross-linking and LasA cleavage of pentaglycine cross-bridges
results in extensive cell lysis and a potent bactericidal effect.
Expression of LasA has been observed in clinical P. aeruginosa
isolates, and the protein itself has been detected in the sputum of
cystic fibrosis patients. These findings suggest that LasA
production by P. aeruginosa may be an important determinant of
vancomycin efficacy against S. aureus during the treatment of
co-infections.
P. aeruginosa Rhamnolipids Increase Tobramycin Uptake and Efficacy
Against S. aureus
[0143] Supernatant from both PAO1 and PA14 overnight cultures
induce a 4- to 8-fold increase in the MIC of tobramycin, similar to
previous reports (Table 1). Furthermore, exogenous addition of HQNO
conferred full protection from tobramycin killing (FIG. 7D).
Paradoxically, treatment with PAO1 or PA14 supernatant led to no
significant induction of tobramycin tolerance in S. aureus (FIG.
D). Strikingly, it was found that PA14 .DELTA.pqsLphzShenC mutant
supernatant in conjunction with tobramycin treatment results in the
rapid eradication of a S. aureus population (FIGS. 4A and 4B).
Further, it was found that PA14 .DELTA.pqsLphzShenC supernatant
reduced the MIC of tobramycin 2-fold relative to the control and 8-
to 16-fold relative to PA14 treated S. aureus cells (Table 1). This
suggests that an unknown factor present in P. aeruginosa
supernatant is responsible for potentiating tobramycin killing.
Heat-inactivation of P. aeruginosa supernatant did not induce
tobramycin tolerance ruling out heat-labile proteins as possible
potentiators of tobramycin killing (FIG. 8A).
[0144] Tobramycin uptake is dependent on PMF. HQNO collapses S.
aureus PMF by inhibiting electron transport, and thus abolishes
tobramycin uptake into the cell. It was hypothesized that a
potentiating agent may be able to induce tobramycin uptake in a
PMF-independent manner P. aeruginosa produces surfactant molecules
called rhamnolipids that inhibit growth of competing Gram-positive
bacteria and increase permeability by interacting with the plasma
membrane. It was hypothesized that by increasing uptake in a
PMF-independent manner, P. aeruginosa rhamnolipids may be
potentiating tobramycin uptake in S. aureus . To investigate this
possibility, a deletion was created in the rhL4 gene in PA14, which
is essential for rhamnolipid biosynthesis. Supernatant from a PA14
.DELTA.rhlA mutant conferred full protection to S. aureus against
tobramycin killing (FIGS. 4A and 4B). Furthermore, during
tobramycin treatment, the exogenous addition of a 50-50 mix of
purified P. aeruginosa mono- and di-rhamnolipids at a concentration
of 30 and 50 .mu.g/ml facilitated the rapid eradication of the S.
aureus population (FIG. 4C). At these concentrations, rhamnolipids
did not display bactericidal activity in the absence of antibiotic
(FIG. 8B). It was found that at concentrations of 10, 30 and 50
.mu.g/ml purified P. aeruginosa rhamnolipids reduced the MIC of
tobramycin in S. aureus 2-, 4-, and 8-fold, respectively, (Table 1)
and led to increased uptake of Texas Red-conjugated tobramycin as
determined by flow cytometry (FIG. 4D). These data show that P.
aeruginosa has the capacity to both positively and negatively
influence S. aureus aminoglycoside uptake through the action of
rhamnolipids and respiratory toxins, respectively. Furthermore, we
demonstrate that low concentrations of purified rhamnolipids
facilitate complete eradication of otherwise tolerant S. aureus
persister populations in the presence of tobramycin. The remarkable
ability of rhamnolipids to potentiate aminoglycoside killing, even
in the absence of PMF, may be of major significance for antibiotic
adjuvant development, and could lead to novel therapies with the
capacity to eradicate recalcitrant S. aureus populations.
The Production of HQNO, LasA and Rhamnolipids is Highly Variable in
P. aeruginosa Clinical Isolates
[0145] P. aeruginosa potentiation and antagonism of antibiotic
activity against S. aureus is complex and multifactorial. 5 P.
aeruginosa isolates taken from CF lung infections and 5 P.
aeruginosa isolates from acute burn wound infections were obtained.
HQNO, LasA and rhamnolipid concentrations in the supernatant of
each isolate were quantified then each isolate's capacity to alter
S. aureus antibiotic susceptibility was examined. As HQNO-mediated
inhibition of S. aureus respiration induces the expression of pfiB
(FIG. 2A), a PpflB::gfp reporter was used to measure relative HQNO
levels in P. aeruginosa supernatants (FIG. 5A). In addition, HQNO
production by these isolates was directly quantified through mass
spectrometry (Table 2). LasA expression was quantified by Western
blot as well as by the capacity of each supernatant to lyse a
culture of heat-killed S. aureus (FIG. 5B). Rhamnolipids were
measured by drop collapse assay, a qualitative measurement of
biosurfactant activity (FIG. 5C). It was found that the production
of each of these factors is highly variable between clinical
isolates (FIGS. 5A-5C). As these molecules have a dramatic impact
on S. aureus antibiotic susceptibility, this variance may be an
important factor in determining the outcome of antibiotic
treatment.
[0146] Interestingly, 8 of 10 isolates were capable of inducing
protection from ciprofloxacin killing and this correlated perfectly
with HQNO levels in the supernatant as well as the induction of our
PpflB::gfp reporter (FIGS. 5A and 5D) (Table 2). Similarly, of the
8 supernatants that stimulated at least a 10-fold potentiation of
vancomycin killing, 6 were positive for LasA, as measured by
Western blot, lytic assay or both (FIGS. 5B and 5E). BC236 and
BC251 potentiated vancomycin killing in the absence of apparent
LasA activity. These strains may produce LasA at levels below the
limit of detection or may potentiate vancomycin through an
unidentified mechanism. Of note, neither of these strains produced
detectable levels of HQNO, which is inhibitory of vancomycin
killing (Table 2).
[0147] CF isolate BC239 was a high producer of HQNO and did not
produce rhamnolipids. In agreement with these findings, supernatant
from this isolate conferred marked protection in S. aureus against
tobramycin killing (FIGS. 5A, 5C, and 5F) (Table 2). Conversely,
supernatant from strain BC249, a burn isolate and the highest
rhamnolipid producer examined, increased killing by tobramycin
approximately 10-fold (FIGS. 5C and 5F).
Discussion
[0148] During such co-infections, P. aeruginosa employs an arsenal
of weaponry to compete with S. aureus . A model is proposed whereby
antibiotic efficacy against S. aureus is determined by interactions
with co-infecting P. aeruginosa (FIG. 6). In support of this, it
was demonstrated that HQNO-mediated inhibition of respiration
forces S. aureus into a low ATP, multidrug tolerant state. It was
further demonstrated that the presence of LasA in P. aeruginosa
supernatant is sufficient to override this protective effect, and
instead potentiates vancomycin-mediated cell lysis and death.
Similarly, though HQNO mediates S. aureus resistance to tobramycin,
it was shown that P. aeruginosa -produced rhamnolipids can negate
this effect and restore or even increase S. aureus susceptibility
to tobramycin killing. Stimulating tobramycin uptake has been
proposed as a way to eradicate persister populations. The present
data suggest that rhamnolipids facilitate penetration of tobramycin
independently of proton-motive force. Exogenous addition of
rhamnolipids to S. aureus cultures resulted in dramatic
potentiation of tobramycin killing, leading to total eradication of
S. aureus persister populations. Further exploitation of LasA and
rhamnolipid-mediated antibiotic potentiation could lead to future
antibiotic adjuvants that facilitate eradication of recalcitrant S.
aureus populations for the treatment of chronic infection.
[0149] It was found that the capacity of clinical P. aeruginosa
isolates to antagonize or potentiate the killing activities of
antibiotics against S. aureus is highly variable and dependent on
the production of antistaphylococcal compounds. The production of
HQNO, LasA and rhamnolipids are regulated by quorum sensing (QS) in
P. aeruginosa. P. aeruginosa frequently accumulates mutations in
genes required for QS during chronic infection. Interestingly, in
support of this, strains from acute burn wound infections appear to
produce higher levels of HQNO, LasA and rhamnolipids, while
isolates from CF lung infections tended to produce less of these
compounds. The diversity of HQNO, LasA and rhamnolipid production
by P. aeruginosa clinical isolates and their impact on S. aureus
suggests that genotypic variation of P. aeruginosa may have a
significant impact on antibiotic susceptibility of S. aureus during
co-infection. It will be interesting to measure the levels of these
antistaphylococcal factors in a larger clinical strain cohort in
future studies.
TABLE-US-00001 TABLE 1 Minimum inhibitory concentrations (MIC) of
S. aureus HG003 +HG003 PAO1 and PA14 PA14 Antibiotic Control
supernatant supernatant .DELTA.pqsLphzShcnC MIC ciprofloxacin
(.mu.g/ml) 0.3 0.3 0.3 -- MIC tobramycin (.mu.g/ml) 0.78 0.78
3.125-6.25 0.39 MIC oxacillin (.mu.g/ml) 0.39 0.39 0.39 -- MIC
vancomycin (.mu.g/ml) 1.25 1.25 1.25 -- 10 .mu.g/ml 30 .mu.g/ml 50
.mu.g/ml Antibiotic Control rhamnolipids rhamnolipids rhamnolipids
MIC tobramycin (.mu.g/ml) 0.78 0.39 0.195 0.0975
TABLE-US-00002 TABLE 2 LC-MS/MS quantification of HQNO production
in P. aeruginosa strains Strain Conc. (.mu.M) PAO1 31.5 PA14 28.3
PA14 .DELTA.pqsL ND BC236 ND BC237 18.9 BC238 13.9 BC239 25.7 BC240
ND BC249 29.0 BC250 28.2 BC251 ND BC252 47.0 BC253 9.8
EXAMPLE 2
Effect of Tobramycin and Rhamnolipids on Biofilms
[0150] For biofilm killing assays, S. aureus was grown in 96-well
polystyrene plates in Tryptic Soy Broth (TSB) overnight. Media was
removed and biofilm formed on the base of the plates was washed 3
times in sterile PBS. Fresh media containing tobramycin alone,
rhamnolipids alone or a combination was added and plates were
incubated overnight. Media was removed. Biofilm was washed 3 times.
Sterile PBS was added to each well and plates were placed in a
sonicating water bath for 10 minutes to remove the biofilm from the
plate. PBS containing solubilized biofilm was then serially diluted
and plated on MHA plates to enumerate survivors. The results are
shown in FIG. 9.
EXAMPLE 3
Effect of Tobramycin and Rhamnolipids on Anaerobic Killing
[0151] For anaerobic killing assays, S. aureus was grown in MHB in
an anaerobic chamber to mid-exponential phase. Tobramycin,
rhamnolipids and a combination of the two were then added to
cultures and the cultures were incubated anaerobically overnight.
Cultures were then centrifuged and cells were washed, serially
diluted and plated for survivors. The results are shown in FIG.
10.
EXAMPLE 4
Effect of Tobramycin and Rhamnolipids on S. aureus
[0152] The ability of rhamnolipids to potentiate killing of S.
aureus by tobramycin was examined. S. aureus HG003 was grown in MHB
for 3 hours 30 minutes and then challenged with tobramycin at 58
.mu.g/ml alone or in combination with 30 .mu.g/ml rhamnolipids. The
combination showed eradication of the population within 5 hours
while the use of tobramycin alone resulted in 10.sup.4 survivors
even after 12 hours (FIG. 11).
EXAMPLE 5
Effect of Tobramycin and Rhamnolipids on Tobramycin Resistant S.
aureus Isolates
[0153] Tobramycin resistant S. aureus isolates were cultured from
the sputum of 6 cystic fibrosis patients. MIC experiments were
performed with tobramycin in MHB. MIC experiments were then
repeated in the presence of 50 .mu.g/ml rhamnolipids. Rhamnolipids
reduced the MIC significantly (FIG. 12).
EXAMPLE 6
Effect of Tobramycin and Rhamnolipids on Low Energy S. aureus
[0154] S. aureus HG003 was grown to late exponential phase, to
which was added tobramycin alone at 58 .mu.g/ml, arsenate alone at
1 mM, arsenate in combination with tobramycin, and arsenate,
tobramycin and rhamnolipids (at 30 .mu.g/ml). cfu/ml were
quantified before addition and after 24 hours, when survivors were
enumerated (FIG. 13).
[0155] It was previously shown that S. aureus reaches an antibiotic
tolerant state by maintaining a low intracellular ATP
concentration. Low ATP can be induced artificially using arsenate.
Under these conditions, tobramycin fails to kill S. aureus .
However, in the presence of rhamnolipids, even this reduction in A
I'P is not protective and the population is reduced by 6 logs over
24 hours. The low energy state is thought to be highly relevant in
vivo where nutrient limitation occurs and cells maintain a far
slower growth rate than in vitro.
EXAMPLE 7
Effect of Tobramycin and Rhamnolipids on CCCP-Treated S. aureus
[0156] S. aureus HG003 was grown to exponential phase, to which was
added tobramycin (58 .mu.g/ml) alone or in combination with
carbonyl cyanide m-chlorophenyl hydrazine (CCCP; 11.1 .mu.g/ml) or
rhamnolipids (30 .mu.g/ml). Survivors were enumerated after 19
hours and 24 hours (FIG. 14).
[0157] CCCP collapses the membrane potential and inhibits
tobramycin uptake and cell death. Even in the presence of CCCP,
rhamnolipids can induce uptake of tobramycin and killing of the S.
aureus population.
EXAMPLE 8
Effect of Protein Synthesis Inhibitors on Tobramycin and
Rhamnolipids-Mediated Killing of S. aureus
[0158] S. aureus HG003 was grown in MHB for 3 hours 30 minutes and
then challenged with tobramycin at 58 .mu.g/ml alone or in
combination with 50 .mu.g/ml of linezolid (added I hour before), 50
.mu.g/ml rhamnolipids or both. Survivors were enumerated at various
timepoints (FIG. 15).
[0159] During conditions where protein synthesis is low or
inhibited, tobramycin fails to kill as is seen in the
tobramycin/linezolid combination experiment. This is important as
an array of physiologically relevant conditions will result in low
protein synthesis rates relative to those seen under ideal
conditions in vitro. Importantly, even in the presence of
linezolid, a combination of tobramycin and rhamnolipids is capable
of inducing cell death, while tobramycin alone fails to kill.
EXAMPLE 9
Effect of Tobramycin and Rhamnolipids on Development of
Tobramycin-Resistant S. aureus
[0160] S. aureus was sub-cultured in increasing concentrations of
tobramycin in the presence or absence of rhamnolipids. MIC
experiments were performed daily, with the bacteria growing in the
highest concentration of antibiotic used as the inoculum for the
next MIC. This procedure selects for the bacteria most capable of
growing in the antibiotic. For 3 separate experiments, high level
resistance to tobramycin was selected for by day 10 and it
increased as high as 100 .mu.g/ml by day 26 (FIG. 16). This high
level resistance is similar to that observed in isolates from
cystic fibrosis patients lungs. Interestingly, in the presence of 3
.mu.g/ml rhamnolipids, resistance does not develop, even after 27
days of sub-culture.
EXAMPLE 10
Effect of Tobramycin and Rhamnolipids on a Small Colony Variant of
S. aureus
[0161] A menD mutant was constructed in S. aureus HG003. This
mutant grows slowly and is resistant to tobramycin due to a
collapse in membrane potential. The mutant was grown to exponential
phase and tobramycin was added alone or in combination with
rhamnolipids at 10 .mu.g/ml or 30 .mu.g/ml (FIG. 17). Rhamnolipids
sensitized the small colony variant to tobramycin and at 30
.mu.g/ml facilitated eradication to limit of detection within 2
hours. SCVs are highly physiologically relevant and very difficult
to kill with conventional antibiotics. These data suggest that
combining antibiotic with rhamnolipids will facilitate rapid
eradication of small colony variants.
[0162] MIC experiments were carried out with the menD mutant in MH
broth. It was found that addition of rhamnolipids at 50 .mu.g/ml
resulted in a 511 fold reduction in MIC of the SCV to tobramycin
(FIG. 18).
EXAMPLE 11
Effect of Rhamnolipids and Other Membrane-Permeabilizing Molecules
on S. aureus
[0163] S. aureus HG003 was grown to exponential phase in a 96 well
plate. Rhamnolipids, palmitoleic acid (Palm50), and glycerol
monolaureate (GML) were added. These concentrations were not lethal
to S. aureus alone. Propidium iodide (PI) was then added to measure
the permeability of the S. aureus membrane (FIG. 19A). PI only
fluoresces when it penetrates the cell and under normal conditions,
PI will not penetrate live bacteria. When the membrane is
compromised, PI can enter. The ability of these permeabilizing
molecules in combination with tobramycin to kill S. aureus over 24
hours was quantitated (FIG. 19B). The data show that other
membrane-permeabilizing surfactants can synergize with tobramycin
to penneabilize S. aureus . An array of other chemicals known to
permeabilize the S. aureus membrane were found to potentiate
aminoglycoside killing of S. aureus.
EXAMPLE 12
Effect of Different Rhamnolipids on Potentiation of Tobramycin
[0164] Various rhamnolipids were added to S. aureus HG003 in
combination with tobramycin at 15.6 .mu.g/ml (equivalent to 20
times the minimum inhibitory concentration). RL90 is a mix of
rhamnolipids containing mono- and di-rhamnolipids with different
carbon chain lengths. RL95D90 is 95% pure rhamnolipids, and 90%
di-rhamnolipid. RLC10-C10 is pure mono-rhamnolipid with 2 CIO
carbon tails. RLC12-C12 is pure mono-rhamnolipid with 2 C12 carbon
tails. All rhamnolipids tested could potentiate tobramycin killing
with the di-rhamnolipid dominant rhamnolipids displaying less
activity than mono-rhamnolipids (FIG. 20).
[0165] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
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