U.S. patent application number 11/260676 was filed with the patent office on 2006-11-23 for methods of reducing microbial resistance to drugs.
This patent application is currently assigned to Trustees of Tufts College. Invention is credited to Stuart B. Levy, Margaret Oethinger.
Application Number | 20060264517 11/260676 |
Document ID | / |
Family ID | 23411689 |
Filed Date | 2006-11-23 |
United States Patent
Application |
20060264517 |
Kind Code |
A1 |
Oethinger; Margaret ; et
al. |
November 23, 2006 |
Methods of reducing microbial resistance to drugs
Abstract
The instant methods and compositions represent an advance in
controlling drug resistance in microbes. AcrAB-like efflux pumps
have been found to control resistance to drugs, even in highly
resistant microbes. Accordingly, methods of treating infection,
methods of screening for inhibitors of AcrAB-like efflux pumps, and
methods of enhancing antimicrobial activity of drugs are provided.
Pharmaceutical composition comprising an inhibitor of an AcrAB-like
efflux pump and an antimicrobial agent are also provided.
Inventors: |
Oethinger; Margaret; (Bad
Oeynhausen, DE) ; Levy; Stuart B.; (Boston,
MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Trustees of Tufts College
Medford
MA
|
Family ID: |
23411689 |
Appl. No.: |
11/260676 |
Filed: |
October 26, 2005 |
Related U.S. Patent Documents
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10629340 |
Jul 28, 2003 |
7026136 |
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11260676 |
Oct 26, 2005 |
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09966835 |
Sep 28, 2001 |
6677133 |
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10629340 |
Jul 28, 2003 |
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09358948 |
Jul 22, 1999 |
6346391 |
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09966835 |
Sep 28, 2001 |
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Current U.S.
Class: |
514/721 ;
514/253.08; 514/312 |
Current CPC
Class: |
A61K 31/496 20130101;
C12Q 1/689 20130101; C12Q 2600/136 20130101; A61P 43/00 20180101;
A61P 31/00 20180101; A61K 45/06 20130101; A61K 31/09 20130101; Y02A
50/481 20180101; A61K 31/47 20130101; C12Q 2600/158 20130101; C12Q
1/18 20130101; Y02A 50/30 20180101; A61K 31/085 20130101; A61P
31/04 20180101; C12Q 2600/156 20130101; A61K 31/09 20130101; A61K
2300/00 20130101; A61K 31/47 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/721 ;
514/253.08; 514/312 |
International
Class: |
A61K 31/496 20060101
A61K031/496; A61K 31/4704 20060101 A61K031/4704; A01N 31/14
20060101 A01N031/14 |
Goverment Interests
GOVERNMENT FUNDING
[0001] This work was funded, at least in part, by a research grant
from the U.S. Public Health Service GM 51661. The government,
therefore, may have certain rights in the invention.
Claims
1. A method of treating an infection caused by a drug resistant
microbe in a subject comprising administering a drug to which the
microbe is resistant and an inhibitor of an AcrAB-like efflux pump
to the subject such that the infection is treated.
2. The method of claim 1, wherein the drug is an antibiotic.
3. The method of claim 2, wherein the antibiotic is a
fluoroquinolone.
4. The method of claim 1, wherein the drug is a non-antibiotic
agent.
5. The method of claim 4, wherein the non-antibiotic agent is
triclosan.
6. The method of claim 1, wherein the inhibitor of an AcrAB-like
efflux pump is administered prophylacticly.
7. The method of claim 1, wherein the inhibitor of an AcrAB-like
efflux pump is administered therapeutically.
8. A method of treating a fluoroquinolone resistant infection in a
subject comprising administering a fluoroquinolone and an inhibitor
of an AcrAB-like efflux pump to the subject to thereby treat a
fluoroquinolone resistant infection.
9. A method of screening for compounds which reduce drug resistance
comprising: contacting a microbe comprising an AcrAB-like efflux
pump with a test compound and a indicator compound and measuring
the effect of the test compound on efflux of the indicator compound
to thereby identify compounds which reduce drug resistance by
inhibiting the activity of an AcrAB efflux pump.
10. The method of claim 9, wherein the microbe is highly drug
resistant.
11. The method of claim 9, wherein the microbial cell is highly
resistant to fluoroquinolones.
12. The method of claim 9, wherein the microbial cell comprises at
least one chromosomal mutation in a drug target gene.
13. The method of claim 12, wherein the mutation is present in a
gene selected from the group consisting of: gyrase, topoisomerase,
and fabI.
14. The method of claim 6, comprising detecting the ability of the
compound to reduce fluoroquinolone resistance in a microbe.
15. A method of screening for compounds which specifically inhibit
the activity of an AcrAB-like efflux pump comprising: i) contacting
a microbe comprising an AcrAB-like efflux pump with a test compound
and an indicator compound; ii) testing the ability of the compound
to inhibit the activity of an AcrAB-like efflux pump; iii) testing
the ability of the compound to inhibit the activity of a non-AcrAB
efflux pump; iv) and identifying compounds which inhibit the
activity of an AcrAB-like efflux pump and non a non-AcrAB-like
efflux pump to thereby identify compounds which specifically block
an AcrAB-like efflux pump.
16. A method of enhancing the antimicrobial activity of a drug
comprising: contacting a microbe that is resistant to one or more
drugs with a drug to which the microbe is resistant and an
inhibitor of an AcrAB-like efflux pump to thereby enhance the
antimicrobial activity of a drug.
17. The method of claim 16 wherein the microbe is contacted with a
compound selected from the group consisting of: cyclohexadine,
quaternary ammonium compounds, pine oil, triclosan, and compound
generally regarded as safe.
18. The method of claim 16, wherein the step of contacting occurs
ex vivo.
19. The method of claim 16, wherein the microbe is contacted with a
non-antibiotic agent and an inhibitor of an AcrAB-like efflux
pump.
20. The method of claim 19, wherein the non-antibiotic agent is
selected from the group consisting of: chlorhexadine, quaternary
ammonium compounds, pine oil, triclosan, and compound generally
regarded as safe (GRAS).
21. A pharmaceutical composition comprising an inhibitor of an
AcrAB-like efflux pump and an antibiotic.
22. The composition of claim 20, further comprising a
pharmaceutically acceptable carrier.
23. The composition of claim 21, wherein the antibiotic is a
fluoroquinolone.
24. The composition of claim 22, wherein the pharmaceutically
acceptable carrier is an inhibitor of the pump.
Description
BACKGROUND OF THE INVENTION
[0002] Different drugs used to inhibit microbial growth act by
inhibiting different targets. For example, the fluoroquinolone
class of antibiotics act by inhibiting bacterial DNA synthesis.
When used in treatment, fluoroquinolones are well absorbed orally,
are found in respiratory secretions in higher concentrations than
in serum and are concentrated inside macrophages. In addition,
fluoroquinolones are well tolerated and have an excellent safety
record in long-term therapy.
[0003] Antibiotic resistance, and in particular resistance to
fluoroquinolones, has become a problem. Fluoroquinolone resistance
in gram negative bacteria is principally caused by mutations
affecting the target proteins of the drugs. In the case of
fluoroquinolones, these targets are DNA gyrase and topoisomerase
IV. In addition, mutations affecting regulatory genes such as marA,
soxS or rob can cause fluoroquinolone resistance (Oethinger et al.
1998. J. Antimicrob. Chemother. 41:111). Mar A is a transcriptional
activator encoded by the marRAB operon involved in multiple
antibiotic resistance (Alekshun et al. (1997) Antimicrob. Agents
Chemother. 41, 2067-2075). The marRAB locus confers resistance to
tetracycline, chloramphenicol, fluoroquinolones, nalidixic acid,
rifampin, penicillin, as well as other compounds. However, marRAB
does not encode a multidrug efflux system. Rather, it controls the
expression of other loci important in directly mediating drug
resistance, e.g., ompF, the gene for outer membrane porin, and the
acrAB genes for the AcrAB efflux proteins.
[0004] AcrAB is a multidrug efflux pump (Nikaido, H. (1996) J.
Bacteriol. 178, 5853-5859; Okusu et al. (1996) J. Bacteriol. 178,
306-308) whose normal physiological role is unknown, although it
may assist in protection of cells against bile salts in the
mammalian small intestine (Thanassi et al. (1997) J. Bacteriol.
179, 2512-2518). The AcrAB operon is upregulated by MarA (Ma et al.
(1995) Mol. Microbiol. 16, 45-55). Mutations in the repressor gene
marR lead to overexpression of marA (Alekshun et al. (1997).
Antimicrob. Agents Chemother. 41, 2067-2075; Cohen et al. (1993) J.
Bacteriol. 175, 1484-492); Seoane et al. (1995) J. Bacteriol. 177,
3414-3419). The soxS gene encodes a MarA homolog (Alekshun et al.
(1997) Antimicrob. Agents Chemother. 41, 2067-2075; Li et al.
(1996) Mol. Microbiol. 20, 937-945; Miller et al. (1996) Mol.
Microbiol. 21, 441-448) which also positively regulates acrAB (Ma
et al. (1996) Mol. Microbiol. 19, 101-112).
[0005] The AcrAB pump primarily controls resistance to large,
lipophilic agents that have difficulty penetrating porin channels,
such as erythromycin, fusidic acid, dyes, and detergents, while
leaving microbes susceptible to small antibiotics that can diffuse
through the channel, e.g., tetracycline, chloramphenicol, and
fluoroquinolones (Nikaido. 1996. J. Bacteriology 178:5853).
Recently, the AcrAB pump has been found to be important in
mediating resistance to other drugs used to control microbial
growth, e.g., non-antibiotic agents such as triclosan (FEMS
Microbiol Lett Sep. 15, 1998; 166: 305-9.
[0006] Microbes often become resistant to antibiotics and/or
non-antibiotic agents. This can occur by the acquisition of genes
encoding enzymes that inactivate the agents, modify the target of
the agent, or result in active efflux of the agent. Enzymes that
inactivate synthetic antibiotics such as quinolones, sulfonamides,
and trimethoprim have not been found. In the case of these
antibiotics and natural products for which inactivating or
modifying enzymes have not emerged, resistance usually arises by
target modifications (Spratt. 1994. Science 264:388). Improved
methods for controlling drug resistance in microbes, in particular
in microbes that are highly resistant to drugs, would be of
tremendous benefit.
SUMMARY OF THE INVENTION
[0007] The present invention is based, at least in part, on the
discovery that inactivation of the AcrAB locus makes even resistant
microbial cells hypersusceptible to antibiotics and non-antibiotic
drugs. Surprisingly, this is true even among highly resistant
microbes which have chromosomal mutations that render them highly
resistant to drugs.
[0008] Accordingly, in one aspect, the invention provides methods
of treating an infection caused by a drug resistant microbe in a
subject by administering a drug to which the microbe is resistant
and an inhibitor of an AcrAB-like efflux pump to the subject such
that the infection is treated.
[0009] In one embodiment, the drug is an antibiotic. In a preferred
embodiment the antibiotic is selected from the group consisting of
a fluoroquinolone and rifampin. In another embodiment, the drug is
a non-antibiotic agent. In another embodiment, the drug is the
non-antibiotic agent, triclosan.
[0010] In one embodiment, the inhibitor of an AcrAB-like efflux
pump is administered prophylacticly. In another embodiment, the
inhibitor of an AcrAB-like efflux pump is administered
therapeutically.
[0011] In another aspect, the invention pertains to a method of
treating a fluoroquinolone resistant infection in a subject
comprising administering a fluoroquinolone and an inhibitor of an
AcrAB-like efflux pump to the subject to thereby treat a
fluoroquinolone resistant infection.
[0012] In another aspect, the invention pertains to a method of
screening for compounds which reduce drug resistance comprising:
contacting a microbe comprising an AcrAB-like efflux pump with a
test compound and a indicator compound and measuring the effect of
the test compound on efflux of the indicator compound to thereby
identify compounds which reduce drug resistance by testing the
ability of the test compound to inhibit the activity of an AcrAB
efflux pump.
[0013] In one embodiment, the microbe is drug resistant. In a
preferred embodiment, the microbial cell is highly drug resistant.
In a more preferred embodiment, the microbe is highly resistant to
fluoroquinolones. In another embodiment, the microbial cell
comprises at least one mutation in a drug target gene. In another
embodiment the microbial cell cmprises at least two mutations in a
drug target gene. In a preferred embodiment, a mutation is present
in a gene selected from the group consisting of: gyrase (gyrA),
topoisomerase (parC), RNA polymerase, and fabI.
[0014] In one embodiment, the subject assay includes detecting the
ability of the compound to reduce fluoroquinolone resistance in a
microbe.
[0015] In another aspect, the invention provides a method of
screening for compounds which specifically inhibit the activity of
an AcrAB-like efflux pump comprising:
[0016] i) contacting a microbe comprising an AcrAB-like efflux pump
with a test compound and an indicator compound;
[0017] ii) testing the ability of the compound to inhibit the
activity of an AcrAB-like efflux pump;
[0018] iii) testing the ability of the compound to inhibit the
activity of a non-AcrAB efflux pump;
[0019] iv) and identifying compounds which inhibit the activity of
an AcrAB-like efflux pump and non a non-AcrAB-like efflux pump to
thereby identify compounds which specifically block an AcrAB-like
efflux pump.
[0020] In yet another aspect, the invention provides a method of
enhancing the antimicrobial activity of a drug comprising:
contacting a microbe that is highly resistant to one or more drugs
with a drug to which the microbe is resistant and an inhibitor of
an AcrAB-like efflux pump to thereby enhance the antimicrobial
activity of a drug.
[0021] In one embodiment, the step of contacting occurs ex vivo. In
one embodiment, the microbe is contacted with a non-antibiotic
agent and an inhibitor of an AcrAB-like efflux pump. In one
embodiment, the non-antibiotic agent is selected from the group
consisting of: cyclohexadine, quaternary ammonium compounds, pine
oil, triclosan, and compound generally regarded as safe (GRAS).
[0022] In another aspect, the invention provides a pharmaceutical
composition comprising an inhibitor of an AcrAB-like efflux pump
and an antibiotic. In one embodiment, the pharmaceutical
composition further comprises a pharmaceutically acceptable
carrier. In a preferred embodiment, the antibiotic is selected from
the group consisting of fluoroquinolone and rifampin.
BRIEF DESCRIPTION OF THE DRAWING
[0023] FIG. 1 shows accumulation of ciprofloxacin (CIP) by
energized cells. [.sup.14C-]ciprofloxacin uptake by
fluoroquinolone-resistant mutants derived in vitro from E. coli-K12
strains AG100 and AG112 was assayed at 30.degree. C. at equilibrium
after addition of 10 .mu.M ciprofloxacin. Cells carried either the
wild-type acrAB gene (solid bars) or an acrAB-deletion (hashed
bars).
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention represents an advance in controlling
resistance to drugs which are substrates of AcrAB-like efflux
pumps. In preferred embodiments, the methods of the invention can
be used to control resistance to antibiotic agents and
non-antibiotic agents. In a particularly preferred embodiment,
drugs which are substrates of AcrAB-like efflux pumps include
antibiotics, e.g., fluoroquinolones or rifampin. The invention
further pertains to AcrAB-like efflux pump inhibitors obtained
using the instant methods and their methods of use. In another
particularly preferred embodiment, drugs include non-antibiotic
agents, e.g., triclosan. The subject methods are effective in
controlling drug resistance even among highly resistant microbes
that bear chromosomal mutations which alter drug target molecules.
For example, analysis of microbial mutants has revealed that
mutations in the fluoroquinolone target gene gyrA, the regulatory
gene marR, and additional, as yet unidentified genes probably
affecting AcrAB-mediated efflux of ciprofloxacin all contributed to
fluoroquinolone resistance. Surprisingly, inactivation of the AcrAB
locus made even highly resistant cells hypersusceptible to
fluoroquinolones and certain other unrelated drugs even among
topoisomerase mutants. These studies indicate that blocking the
function of AcrAB-like efflux pumps reduces even high-level drug
resistance.
[0025] Accordingly, the invention provides, inter alia, methods of
screening for compounds which reduce drug resistance, in particular
to antibiotics and non-antibiotic agents such as fluoroquinolones
and triclosan, and methods of screening for compounds that
specifically block an AcrAB-like efflux pump. In addition, the
invention provides methods of enhancing the antimicrobial activity
of a drug and methods of treating infection.
[0026] Before further description of the invention, certain terms
employed in the specification, examples and appended claims are,
for convenience, collected here.
I. Definitions
[0027] As used herein the term "infection" includes the presence of
a microbe in or on a subject which, if its growth were inhibited,
would result in a benefit to the subject. As such, the term
"infection" in addition to referring to the presence of pathogens
also includes normal flora which is not desirable, e.g., on the
skin of a burn patient or in the gastrointestinal tract of an
immunocompromised patient. As used herein, the term "treating"
refers to the administration of a compound to a subject, for
prophylactic and/or therapeutic purposes. The term "administration"
includes delivery to a subject, e.g., by any appropriate method
which serves to deliver the drug to the site of the infection.
Administration of the drug can be, e.g., oral, intravenous, or
topical.
[0028] As used herein, the term "drug" includes compounds which are
substrates of AcrAB-like efflux pumps. The term "drug" includes
compounds which reduce the growth of a microbe e.g., which reduce
the ability of a microbe to produce infection in a host, or which
reduce the ability of a microbe to multiply or remain infective in
the environment. Such drugs include antibiotic agents and
non-antibiotic agents. The term "drug" includes antiinfective
compounds which are static or cidal for microbes, e.g., an
antimicrobial compound which inhibits the growth and/or viability
of a microbe. Preferred antiinfective compounds increase the
susceptibility of microbes to antibiotics or decrease the
infectivity or virulence of a microbe. Substrates of the AcrAB-like
efflux pumps can be readily identified using methods known in the
art and described in further detail herein. For example, putative
drugs which are substrates of AcrAB-like efflux pumps can be
labeled, e.g., radioactively, and their export from a microbial
cell tested in microbial cells which possess AcrAB-like efflux
pumps and in microbial cells which lack AcrAB-like efflux pumps.
Those agents which are present at a higher intracellular
concentration in microbes that lack such pumps than in microbes
that possess such pumps are drugs which are substrates of the
pump.
[0029] The term "drug" includes the antimicrobial agents to which
the Mar phenotype has been shown to mediate resistance and, as
such, includes disinfectants, antiseptics, and surface delivered
compounds. For example, antibiotics, biocides, or other type of
antibacterial compounds, including agents which induce oxidative
stress agents, and organic solvents are included in this term. The
term "drug" also includes biocidal agents. The term "biocidal" is
art recognized and includes an agent that those ordinarily skilled
in the art prior to the present invention believed would kill a
cell "non-specifically," or a broad spectrum agent whose mechanism
of action is unknown, e.g., prior to the present invention, one of
ordinary skill in the art would not have expected the agent to be
target-specific. Examples of biocidal agents include paraben,
chlorbutanol, phenol, alkylating agents such as ethylene oxide and
formaldehyde, halides, mercurials and other heavy metals,
detergents, acids, alkalis, and chlorhexidine. Other biocidal
agents include: triclosan, pine oil, quaternary amine compounds
such as alkyl dimethyl benzyl ammonium chloride, chloroxylol,
chlorhexidine, cyclohexidine, triclocarbon, and disinfectants. The
term "bactericidal" refers to an agent that can kill a bacterium;
"bacteriostatic" refers to an agent that inhibits the growth of a
bacterium.
[0030] The term "antibiotic" is art recognized and includes
antimicrobial agents synthesized by an organism in nature and
isolated from this natural source, and chemically synthesized
drugs. The term includes but is not limited to: polyether ionophore
such as monensin and nigericin; macrolide antibiotics such as
erythromycin and tylosin; aminoglycoside antibiotics such as
streptomycin and kanamycin; .beta.-lactam antibiotics such as
penicillin and cephalosporin; and polypeptide antibiotics such as
subtilisin and neosporin. Semi-synthetic derivatives of
antibiotics, and antibiotics produced by chemical methods are also
encompassed by this term. Chemically-derived antimicrobial agents
such as isoniazid, trimethoprim, quinolones, fluoroquinolones and
sulfa drugs are considered antibacterial drugs, and the term
antibiotic includes these. It is within the scope of the screens of
the present invention to include compounds derived from natural
products and compounds that are chemically synthesized.
[0031] In contrast to the term "biocidal," an antibiotic or an
"anti-microbial drug approved for human use" is considered to have
a specific molecular target in a microbial cell. Preferably a
microbial target of a therapeutic agent is sufficiently different
from its physiological counterpart in a subject in need of
treatment that the antibiotic or drug has minimal adverse effects
on the subject.
[0032] The phrase "non-antibiotic agent" includes substrates of an
acrAB-like efflux pump which are not art recognized as being
antibiotics. Exemplary non-antibiotic agents include, e.g.,
biocides, disinfectants or antiinfectives. Non antibiotic agents
also include substrates of an acrAB-like efflux pump which are
incorporated into consumer goods, e.g., for topical use on a
subject or as cleaning products.
[0033] As used herein, the term "fluoroquinolone" includes
quinolones substituted with at least one fluorine atom. Preferred
fluoroquinolones include compounds with the carbonyl at the 4
position. Preferred positions for fluorine substitution include the
5, 6, and 7 positions. Derivatives include compounds with
additional substituents such as, although not limited to, NR'R'',
CN, NO.sub.2, F, Cl, Br, I, CF.sub.3, CCl.sub.3, CHF.sub.2,
CHCl.sub.2, CONR'R'', S(O)NR'R'', CHO, OCF.sub.3, OCCl.sub.3,
SCF.sub.3, SCCl.sub.3, COR', CO.sub.2R', and OR' and wherein R' and
R'' are each independently hydrogen, C.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4 alkynyl or optionally
substituted cyclic or heterocyclic groups. The term fluoroquinolone
also encompasses compounds with heterocyclic substitutions at the
seven position, such as naphthyridinones. Preferred substituents
include piperazinyl groups and other heterocyclic groups, ,
carboxylic acid groups and substituted or unsubstituted alkyl
groups. One example of a preferred fluoroquinolone is shown below,
wherein X is CH or N or CR.sub.6 and where R.sub.1 is lower alkyl
including cycloalkyl groups, or optionally substituted with
halogens; R.sub.2, R.sub.3, and R.sub.4 are each independently
substituted or unsubstituted lower alkyl or hydrogen; R.sub.5 is
fluorine, hydrogen or amino; and R.sub.6 is hydrogen or fluorine.
##STR1##
[0034] As used herein, the term "multiple drug resistance (MDR)"
includes resistance to both antibiotic and non-antibiotic
compounds. MDR results from the increased transcription of a
chromosomal or plasmid encoded genetic locus in an organism, e.g.,
a marRAB locus, that results in the ability of the organism to
minimize the toxic effects of a compound to which it has been
exposed, as well as to other non-related compounds, e.g., by
stimulating an efflux pump(s) or microbiological catabolic or
metabolic processes. As used herein, the phrase "microbes which are
resistant to drugs or drug resistant microbes" includes microbes
that are characterized by a mutation in a target gene or by
increased transcription of a genetic locus that affects drug
resistance, e.g., an efflux pump gene.
[0035] As used herein, the phrase "microbes which are resistant to
drugs or drug resistant microbes" includes microbes that are
characterized by mutations in a gene that is the target of a
drug.
[0036] As used herein, the phrase "microbes which are highly
resistant to drugs or highly drug resistant microbes" includes
microbes that are characterized by mutations in multiple (i.e.,
more than one) gene that affects drug resistance. Preferably, a
microbe that is highly resistant to drugs is characterized by at
least two of the following three traits: (1) it comprises at least
one mutation in a gene encoding a drug target that renders the
microbe resistant to one or more drugs (e.g., a gyrase, fabI or
topoisomerase mutation); (2) it comprises a second mutation (to the
same gene or a different gene than in (1)) that increases drug
resistance; and (3) it has increased expression of at least one
efflux pump (e.g., as a result of increased transcription of the
mar locus). The term "mutation" includes an alteration (e.g., a
substitution, deletion, or insertion) of at least one nucleotide in
the sequence of a nucleic acid molecule (either chromosomal or
episomal) in a microbe which is capable of influencing drug
resistance. Such a mutation can result, e.g., in altered gene
regulation in the microbe or in the expression of an altered
polypeptide. Preferably, such mutations are in genes which encode
the target of the drug to which the microbe is resistant.
[0037] In one embodiment, the drug is an antibiotic. In a preferred
embodiment, the drug is a fluoroquinolone.
[0038] In another embodiment, the drug is a non-antibiotic agent,
e.g., triclosan, pine oil, quaternary amine compounds such as alkyl
dimethyl benzyl ammonium chloride, chloroxylol, triclocarbon, or a
disinfectant, described in further detail herein. In a particularly
preferred embodiment, the drug is a substrate of an acrAB-like
efflux pump which is not a non-antibiotic agent.
[0039] Microbes that are highly resistant to drugs are more
resistant to drugs than microbes that that are characterized by
only one of the preceding traits. In general, antibiotics, when
tested for their effect on the growth of such highly resistant
microbes, will yield a minimal inhibitory concentration (MIC) from
between about 2-fold to more than 100-fold higher than that
observed for a microbe that is characterized by only one of the
above traits or a microbe that is multiply antibiotic resistant,
but not highly resistant to drugs.
[0040] As used herein the term "drug target" includes molecules
which are acted on by drugs and which, in a non-resistant microbe,
are altered such that they do not retain their normal function and
the growth of the microbe is inhibited. For example, exemplary drug
targets include: the DNA gyrase and topoisomerase molecules, which
are targets of fluoroquinolone antibiotics; RNA polymerase, which
is a target of rifampin; and FabI, which is a target of triclosan
(Heath R J, e.t. al. 1999. J. Biol Chem; 274; Levy C W et al. 1999.
Nature 398: 383-4; McMurry L M; et al. 1998. Nature. 394: 531). In
one embodiment, a drug target is not FabI.
[0041] As used herein the term "AcrAB-like efflux pump" includes
efflux pumps that have homology with the AcrAB efflux pump of E.
coli. The AcrAB pump is a resistance, nodulation, and division
(RND)-type pump. RND pumps have 12 transmembrane helices. The acrA
and acrB genes have been cloned and sequenced (Ma et al. 1993. J.
Bacteriol. 175:6229). The sequences of AcrAB in E. coli are
deposited as GenBank accession number U00734. The AcrAB genes have
other homologs in E. coli, as well as homologs in other species of
bacteria. For example, homologs of the AcrAB efflux pump have been
identified in Haemophilus influenzae, (Sanchez et al. 1997. J.
Bacteriol. 179:6855) and in Salmonella. typhimurium (Nikaido et al.
1998. J. Bacteriol. 180:4686). Exemplary homologues of AcrAB
include: MtrCD, MexAB-OprM, MexCD-OprJ, MexEF-OprN, and YhiUV. Such
homologs can be readily identified by one of ordinary skill in the
art based on shared homology and structure with the AcrAB pump
and/or based on similarities in the compounds which they export.
Isolation of novel AcrAB-like efflux pumps from other microbes can
be carried out using techniques which are known in the art, e.g.,
nucleic acid hybridization and functional cloning.
[0042] As used herein the term "AcrAB-like efflux pump inhibitor"
refers to a compound which interferes with the ability of an
AcrAB-like efflux pump to export a compound which it is normally
capable of exporting in the absence of such an inhibitor. Such
inhibitors can inhibit the activity of an AcrAB-like efflux pump
directly, e.g., by blocking the pump, or indirectly, e.g., by
reducing transcription of acrA-like and/or acrB-like genes.
Inhibitors of AcrAB-like efflux pumps can inhibit the growth of
resistant and/or highly resistant microbes which used alone, or
they may potentiate the activity of a drug to which the microbe is
resistant.
[0043] As used herein the term "non-AcrAB-like efflux pump"
includes efflux pumps which are not related to the AcrAB efflux
pump of E. coli. Such pumps include, e.g., major facilitator pumps,
membrane fusion proteins, and ABC (ATP-binding cassette) pumps.
[0044] As used herein the term "growth" in reference to the growth
of a microbe includes the reproduction or population expansion of
the microbe, e.g., increase in numbers rather than increase in
size. The term also includes maintenance of on-going metabolic
processes of a microbe, e.g., those processes that keep the cell
alive when the cell is not dividing.
[0045] As used herein the term "reporter gene" includes any gene
which encodes an easily detectable product which gene is operably
linked to a promoter. By operably linked it is meant that under
appropriate conditions an RNA polymerase may bind to the promoter
of the regulatory region and proceed to transcribe the nucleotide
sequence of the reporter gene. In preferred embodiments, a reporter
gene construct consists of a promoter linked to a reporter gene. In
certain embodiments, however, it may be desirable to include other
sequences, e.g., transcriptional regulatory sequences, in the
reporter gene construct. For example, modulation of the activity of
the promoter may be affected by altering the RNA polymerase binding
to the promoter region, or, alternatively, by interfering with
initiation of transcription or elongation of the mRNA. Thus,
sequences which are herein collectively referred to as
transcriptional regulatory elements or sequences may also be
included in the reporter gene construct. In addition, the construct
may include sequences of nucleotides that alter translation of the
resulting mRNA, thereby altering the amount of reporter gene
product.
[0046] As used herein the term "test compound" includes reagents
tested using the assays of the invention to determine whether they
modulate an AcrAB-like efflux pump activity. More than one test
compound, e.g., a plurality of test compounds, can be tested at the
same time for their ability to modulate the activity of an
AcrAB-like efflux pump in a screening assay.
[0047] Compounds that can be tested in the subject assays include
antibiotic and non-antibiotic compounds. Exemplary test compounds
which can be screened for activity include, but are not limited to,
peptides, non-peptidic compounds, nucleic acids, carbohydrates,
small organic molecules (e.g., polyketides), and natural product
extract libraries. The term "non-peptidic compound" is intended to
encompass compounds that are comprised, at least in part, of
molecular structures different from naturally-occurring L-amino
acid residues linked by natural peptide bonds. However,
"non-peptidic compounds" are intended to include compounds
composed, in whole or in part, of peptidomimetic structures, such
as D-amino acids, non-naturally-occurring L-amino acids, modified
peptide backbones and the like, as well as compounds that are
composed, in whole or in part, of molecular structures unrelated to
naturally-occurring L-amino acid residues linked by natural peptide
bonds. "Non-peptidic compounds" also are intended to include
natural products.
[0048] As used herein the phrase "indicator compound" includes
compounds which are normally exported by an AcrAB-like efflux pump.
Indicator compounds are used as markers of AcrAB-like efflux pump
activity in order to determine the effect of a test compound on the
activity of an AcrAB-like efflux pump. Exemplary indicator
compounds include, e.g., antibiotics and dyes.
II. Inhibitors of AcrAB-Like Efflux Pumps
[0049] Known inhibitors of efflux pumps can be used in the methods
and compositions of the invention. Exemplary inhibitors have been
described previously in PCT published patent application W096/33285
(including L-phenylalanyl-L-arginyl-.beta.-naphthylamide). Methods
for testing compounds for efflux pump inhibition are also described
therein. Other useful inhibitors include ethanol (concentrations of
about 4%), methanol, hexane and minocycline. Still other inhibitors
include antisense nucleic acids and ribozymes directed against the
gene(s) encoding the efflux pump. Characteristics of other efflux
pump inhibitors are described, e.g., in WO 96/33285.
[0050] Other exemplary AcrAB-like efflux pump inhibitors include,
e.g., antisense nucleic acids which bind to AcrAB genes and prevent
transcription or translation thereof Antibodies which bind efflux
pumps or proteins which regulate the expression of efflux pumps are
another class of inhibitors. Still other inhibitors include genes
which repress expression of the efflux pumps or regulatory loci
(such as marR) which regulate expression of efflux pumps.
Increasing the amount of such genes or the expression products
thereof can reduce the expression of efflux pumps in microbes.
[0051] As mentioned above, the invention embraces antisense nucleic
acids, including oligonucleotides, that selectively bind to a
nucleic acid molecule encoding an efflux pump (e.g. acrA) or a
molecule which regulates expression of an efflux pump (e.g. marA,
rob or soxS). As used herein, the term "antisense oligonucleotide"
or "antisense molecule" describes an oligonucleotide that is an
oligoribonucleotide, oligodeoxyribonucleotide, modified
oligoribonucleotide, or modified oligodeoxyribonucleotide which
hybridizes under physiological conditions to DNA comprising a
particular gene or to an RNA transcript of that gene and, thereby,
inhibits the transcription of that gene and/or the translation of
that RNA. The antisense molecules are designed so as to interfere
with transcription or translation of a target gene upon
hybridization with the target gene or transcript. Those skilled in
the art will recognize that the exact length of the antisense
oligonucleotide and its degree of complementarity with its target
will depend upon the specific target selected, including the
sequence of the target and the particular bases which comprise that
sequence. It is preferred that the antisense oligonucleotide be
constructed and arranged so as to bind selectively with the target
under physiological conditions, i.e., to hybridize substantially
more strongly to the target sequence than to any other sequence in
the target cell under physiological conditions. Based upon the
nucleic acid sequence of a gene of interest, one of skill in the
art can easily choose and synthesize any of a number of appropriate
antisense molecules for use in accordance with the present
invention. In order to be sufficiently selective and potent for
inhibition, such antisense oligonucleotides should comprise at
least 10 and, more preferably, at least 15 consecutive bases which
are complementary to the target, although in certain cases modified
oligonucleotides as short as 7 bases in length have been used
successfully as antisense oligonucleotides (Wagner et al., Nature
Biotechnol. 14:840-844, 1996). Most preferably, the antisense
oligonucleotides comprise a complementary sequence of 20-30 bases.
Although oligonucleotides may be chosen which are antisense to any
region of the gene or RNA transcripts, in preferred embodiments the
antisense oligonucleotides correspond to N-terminal or 5' upstream
sites such as translation initiation, transcription initiation or
promoter sites. In addition, 3'-untranslated regions may be
targeted. In addition, the antisense is targeted, preferably, to
sites in which RNA secondary structure is not expected and at which
proteins are not expected to bind.
[0052] In embodiment, the antisense oligonucleotides of the
invention may be composed of "natural," i.e., unmodified
deoxyribonucleotides, ribonucleotides, or any combination thereof.
That is, the 5' end of one native nucleotide and the 3' end of
another native nucleotide may be covalently linked, as in natural
systems, via a phosphodiester internucleoside linkage. These
oligonucleotides may be prepared by standard methods which may be
carried out manually or by an automated synthesizer. They also may
be produced recombinantly by vectors.
[0053] In preferred embodiments, however, the antisense
oligonucleotides of the invention also may include "modified"
oligonucleotides. That is, the oligonucleotides may be modified in
a number of ways as compared to naturally occurring
oligonucleotides which do not prevent them from hybridizing to
their target but which enhance their stability or targeting or
which otherwise enhance their therapeutic effectiveness.
[0054] The term "modified oligonucleotide" as used herein describes
an oligonucleotide in which (1) at least two of its nucleotides are
covalently linked via a synthetic internucleoside linkage (i.e., a
linkage other than a phosphodiester linkage between the 5' end of
one nucleotide and the 3' end of another nucleotide) and/or (2) a
chemical group not normally associated with nucleic acids has been
covalently attached to the oligonucleotide. Preferred synthetic
internucleoside linkages are phosphorothioates, alkylphosphonates,
phosphorodithioates, phosphate esters, alkylphosphonothioates,
phosphoramidates, carbamates, carbonates, phosphate triesters,
acetamidates, carboxymethyl esters and peptides.
[0055] The term "modified oligonucleotide" also encompasses
oligonucleotides with a covalently modified base and/or sugar. For
example, modified oligonucleotides include oligonucleotides having
backbone sugars which are covalently attached to low molecular
weight organic groups other than a hydroxyl group at the 3'
position and other than a phosphate group at the 5' position. Thus
modified oligonucleotides may include a 2'-O-alkylated ribose
group. In addition, modified oligonucleotides may include sugars
such as arabinose instead of ribose. The present invention, thus,
provides preparations containing modified antisense molecules that
are complementary to and can hybridize with, under physiological
conditions, nucleic acids encoding mar/sox/rob or efflux pump
polypeptides, together with one or more carriers.
[0056] As described above, the invention further embraces the use
of antibodies or fragments of antibodies having the ability to
selectively bind to efflux pumps, as well as polypeptides which
regulate the expression of efflux pumps. The term "antibody"
includes polyclonal and monoclonal antibodies, or fragments
thereof, prepared according to conventional methodology.
[0057] Accordingly, in another embodiment, antibodies to AcrAB-like
efflux pumps can be used as efflux pump inhibitors. Polyclonal
anti-efflux pump antibodies can be prepared as described above by
immunizing a suitable subject with an immunogen derived from an
AcrAB-like efflux pump. The anti-efflux pump antibody titer in the
immunized subject can be monitored over time by standard
techniques, such as with an enzyme linked immunosorbent assay
(ELISA) using immobilized efflux pump. If desired, the antibody
molecules directed against efflux pump can be isolated from the
mammal (e.g., from the blood) and further purified by well known
techniques, such as protein A chromatography to obtain the IgG
fraction. At an appropriate time after immunization, e.g., when the
anti-efflux pump antibody titers are highest, antibody-producing
cells can be obtained from the subject and used to prepare
monoclonal antibodies by standard techniques, such as the hybridoma
technique originally described by Kohler and Milstein (1975, Nature
256:495-497) (see also, Brown et al. (1981) J. Immunol 127:539-46;
Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et al. (1976) PNAS
76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the
more recent human B cell hybridoma technique (Kozbor et al. (1983)
Immunol Today 4:72), the EBV-hybridoma technique (Cole et al.
(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96) or trioma techniques. The technology for producing
monoclonal antibody hybridomas is well known (see generally R. H.
Kenneth, in Monoclonal Antibodies: A New Dimension In Biological
Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A.
Lemer (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al.
(1977) Somatic Cell Genet., 3:231-36). Briefly, an immortal cell
line (typically a myeloma) is fused to lymphocytes (typically
splenocytes) from a mammal immunized with a efflux pump immunogen
as described above, and the culture supernatants of the resulting
hybridoma cells are screened to identify a hybridoma producing a
monoclonal antibody that binds specifically to an AcrAB-like efflux
pump.
[0058] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-efflux pump monoclonal antibody (see,
e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al.
Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited
supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the
ordinary skilled worker will appreciate that there are many
variations of such methods which also would be useful. Typically,
the immortal cell line (e.g., a myeloma cell line) is derived from
the same mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with an immunogenic preparation of the present invention with an
immortalized mouse cell line. Preferred immortal cell lines are
mouse myeloma cell lines that are sensitive to culture medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Any of a number of myeloma cell lines may be used as a fusion
partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are
available from the American Type Culture Collection (ATCC),
Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are
fused to mouse splenocytes using polyethylene glycol ("PEG").
Hybridoma cells resulting from the fusion are then selected using
HAT medium, which kills unfused and unproductively fused myeloma
cells (unfused splenocytes die after several days because they are
not transformed). Hybridoma cells producing a monoclonal antibody
of the invention are detected by screening the hybridoma culture
supernatants for antibodies that bind efflux pump, e.g., using a
standard ELISA assay.
[0059] As an alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-efflux pump antibody can be
identified and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with efflux pump to thereby isolate immunoglobulin library members
that bind efflux pumps. Kits for generating and screening phage
display libraries are commercially available (e.g., the Pharmacia
Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the
Stratagene SurfZAP.TM. Phage Display Kit, Catalog No. 240612).
Additionally, examples of methods and reagents particularly
amenable for use in generating and screening antibody display
library can be found in, for example, Ladner et al. U.S. Pat. No.
5,223,409; Kang et al. International Publication No. WO 92/18619;
Dower et al. International Publication No. WO 91/17271; Winter et
al. International Publication WO 92/20791; Markland et al.
International Publication No. WO 92/15679; Breitling et al.
International Publication WO 93/01288; McCafferty et al.
International Publication No. WO 92/01047; Garrard et al.
International Publication No. WO 92/09690; Ladner et al.
International Publication No. WO 90/02809; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J
Mol Biol 226:889-896; Clarkson et al. (1991) Nature 352:624-628;
Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)
Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res
19:4133-4137; Barbas et al. (1991) PNAS 88:7978-7982; and
McCafferty et al. Nature (1990) 348:552-554.
[0060] Additionally, recombinant anti-efflux pump antibodies, such
as chimeric and humanized monoclonal antibodies, comprising both
human and non-human portions, which can be made using standard
recombinant DNA techniques, are within the scope of the invention.
Such chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in Robinson et al. International Patent
Publication PCT/US86/02269; Akira, et al. European Patent
Application 184,187; Taniguchi, M., European Patent Application
171,496; Morrison et al. European Patent Application 173,494;
Neuberger et al. PCT Application WO 86/01533; Cabilly et al. U.S.
Pat. No. 4,816,567; Cabilly et al. European Patent Application
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) PNAS 84:3439-3443; Liu et al. (1987) J. Immunol.
139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al.
(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature
314:446-449; and Shaw et al. (1988) J. Natl Cancer Inst.
80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et
al. (1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539;
Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988)
Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
III. Methods of Screening for Novel Inhibitors
[0061] In one aspect, the invention provides a method for screening
for an inhibitor of an AcrAB-like efflux pump. In this method,
microbes expressing an AcrAB-like efflux pump are contacted with a
test compound and a indicator compound. The test compound is a
compound to be tested for its ability to inhibit an AcrAB-like
efflux pump. An indicator compound is one which is normally
exported by the AcrAB-like efflux pump. Using the subject methods
the ability of a test compound to inhibit the activity of an
AcrAB-like efflux pump is demonstrated by determining whether the
intracellular concentration of the indicator compound (e.g., a
fluoroquinolone or a dye) is elevated in the presence of the test
compound. If the intracellular concentration of the indicator
compound is increased in the presence of the test compound as
compared to the intracellular concentration in the absence of the
test compound, then the test compound can be identified as an
inhibitor of an AcrAB-like efflux pump. Thus, one can determine
whether or not the test compound is an inhibitor of an AcrAB-like
efflux pump by showing that the test compound affects the ability
of an AcrAB-like efflux pump present in the microbe to export the
indicator compound.
[0062] The "intracellular concentration" of an indicator compound
includes the concentration of the indicator compound inside the
outermost membrane of the microbe. The outermost membrane of the
microbe can be, e.g., a cytoplasmic membrane. In the case of
Gram-negative bacteria, the relevant "intracellular concentration"
is the concentration in the cellular space in which the indicator
compound localizes, e.g., the cellular space which contains a
target of the indicator compound.
[0063] Inhibitors identified using the subject methods can act
directly on an AcrAB-like pump, e.g., by steric inhibition, or can
act indirectly, e.g., influencing a more distal event, e.g., by
modulating transcription of genes involved in the expression of the
pump.
[0064] In one embodiment, the method comprises detecting the
ability of the compound to reduce fluoroquinolone resistance in a
microbe. For example, in one embodiment, the indicator compound
comprises a fluoroquinolone and the effect of the test compound on
the intracellular concentration of fluoroquinolone in the microbe
is measured. In one embodiment, an increase in the intracellular
concentration of fluoroquinolone can be measured directly, e.g., in
an extract of microbial cells. For example, accumulation of a
radiolabelled fluoroquinolone, e.g., [.sup.14C-]ciprofloxacin can
be determined using standard techniques. For instance, microbes can
be contacted with a radiolabelled fluoroquinolone as an indicator
composition in the presence and absence of a test compound. The
concentration of the fluoroquinolone inside the cells can be
measured at equilibrium by harvesting cells from the two groups
(with and without test compound) and cell associated radioactivity
measured with a liquid scintillation counter. In another
embodiment, an increase in the intracellular concentration of
fluoroquinolone can be measured indirectly, e.g., by a showing that
a given concentration of fluoroquinolone when contacted with the
microbe is sufficient to inhibit the growth of the microbe in the
presence of the test compound, but not in the absence of the test
compound.
[0065] In one embodiment of the subject assays, the step of
determining whether the intracellular concentration of the
indicator compound is elevated is accomplished by measuring a
decrease in the minimal inhibitory concentration (MIC) of the
indicator compound. Such an assay can be performed using a standard
methods, e.g., an antibiotic disc assay.
[0066] In another embodiment, measurement of the intracellular
concentration of an indicator compound can be facilitated by using
an indicator compound which is readily detectable by spectroscopic
means. Such a compound may be, for example, a dye, e.g., a basic
dye, or a fluorophore. Exemplary indicator compounds include:
acridine, ethidium bromode, gentian violet, malachite green,
methylene blue, beenzyn viologen, bromothymol blue, toluidine blue,
methylene blue, rose bengal, alcyan blue, ruthenium red, fast
green, aniline blue, xylene cyanol, bromophenol blue, coomassie
blue, bormocresol purple, bromocresol green, trypan blue, and
phenol red.
[0067] In such an assay, the effect of the test compound on the
ability of the cell to export the indicator compound can be
measured spectroscopically. For example, the intracellular
concentration of the dye or fluorophore can be determined
indirectly, by determining the concentration of the indicator
compound in the suspension medium or by determining the
concentration of the indicator compound in the cells. This can be
done, e.g., by extracting the indicator compound from the cells or
by visual inspection of the cells themselves.
[0068] In another embodiment, the presence of an indicator compound
in a microbe can be detected using a reporter gene which is
sensitive to the presence of the indicator compound. Exemplary
reporter genes are known in the art. For example, a reporter gene
can provide a colorometric read out or an enzymatic read out of the
presence of an indicator compound. In yet another embodiment, a
reporter gene whose expression is inducible by the presence of a
drug in a microbe can be used. For example, a microbe can be grown
in the presence of a drug with and without a putative AcrAB-like
efflux pump inhibitor. In cells in which the efflux pump is
inhibited, the concentration of the drug will be increased and the
reporter gene construct will be expressed. By this method,
AcrAB-like efflux pump inhibitors are identified by their ability
to inhibit the export rate of the drug and, thus, to induce
reporter gene expression.
[0069] In another embodiment, a primary screening assay is used in
which an indicator compound which does not comprise the drug of
interest, e.g., a fluoroquinolone or triclosan is employed. In one
embodiment, upon the identification of a test compound that
increases the intracellular concentration of the test compound, a
secondary screening assay is performed in which the effect of the
same test compound on resistance to the drug of interest, e.g.,
fluoroquinolone resistance, is measured.
[0070] In another aspect, the invention provides a method of
screening for compounds which specifically block an AcrAB-like
efflux pump. In one embodiment the method involves contacting a
microbe comprising an AcrAB-like efflux pump with a test compound
and a indicator compound. The test compound is then tested for its
ability to block an AcrAB-like efflux pump as described supra. The
specificity of compounds which are identified as candidate AcrAB
inhibitory agents can then be tested for their ability to block a
non-AcrAB efflux pump. Compounds which block an AcrAB-like efflux
pump and not a non-AcrAB efflux pump can be identified as compounds
that specifically block an AcrAB-like efflux pump.
[0071] In one embodiment, the use of a compound which is identified
as an inhibitor of an AcrAB-like efflux pump in the subject methods
prevents the development of a drug resistant microbe or of a highly
drug resistant microbe from a drug resistant microbe.
IV. Methods of Enhancing the Antimicrobial Activity of a Drug
[0072] In one aspect the invention pertains to methods of enhancing
the antimicrobial activity of a drug by contacting a microbe that
is resistant to one or more drugs with a drug to which the microbe
is resistant and an inhibitor of an AcrAB-like efflux pump. In one
embodiment, the microbe is contacted with the drug and the
inhibitor of the AcrAB-like efflux pump ex vivo. This method can be
used, e.g., in disinfecting surfaces to prevent the spread of
infection or in cleaning surfaces which are fouled by microbial
growth. Preferably, the drug used to contact the microbe is a
non-antibiotic drug. In a preferred embodiment, the microbe is
contacted with triclosan and an inhibitor of an AcrAB-like efflux
pump.
[0073] In one embodiment, an AcrAB-like efflux pump inhibitor and a
drug can be combined in a disinfectant, e.g., a cleaning product or
a household product for contacting with resistant microbes.
Exemplary cleaning products can be used topically on a subject
(e.g., as soaps or lotions) or can be used for cleaning surfaces.
In one embodiment, an AcrAB-like efflux pump inhibitor is itself a
disinfectant.
V. Methods of Treating Microbial Infections
[0074] In one aspect the invention provides a method of treating a
drug resistant infection in a subject comprising administering a
drug to which a microbe is resistant and an inhibitor of an
AcrAB-like efflux pump to the subject. As used herein the term
"administration" includes contacting a drug with a subject, e.g. in
vivo and/or in vitro. Thus, an efflux pump inhibitor and a drug can
be administered for in vivo treatment or can be used topically,
e.g., on skin or the eyes.
[0075] In one embodiment, the drug is an antibiotic, e.g., a
fluoroquinolone. In another embodiment, the drug is a
non-antibiotic composition, e.g., triclosan. In another embodiment
the infection to be treated is one normally treated with a
non-antibiotic composition. In another embodiment, the infection to
be treated is not one normally treated with a non-antibiotic
composition.
[0076] In one embodiment of the treatment method, the efflux pump
inhibitor and the drug are administered separately to the subject.
In another embodiment, the efflux pump inhibitor and the drug are
administered simultaneously. In one embodiment, the simultaneous
administration of the drug and the AcrAB-like efflux pump inhibitor
is facilitated by the administration of a pharmaceutical
composition comprising both an efflux pump inhibitor and a drug to
which the microbe is resistant.
[0077] The amount of efflux pump inhibitor to be administered to a
subject is a therapeutically effective amount, e.g., for an efflux
pump inhibitor, an amount sufficient to reduce efflux pump
activity. The dosage of efflux pump inhibitor to be administered to
a subject that would benefit from treatment with a drug, e.g. a
patient having an infection with a microbe, can readily be
determined by one of ordinary skill in the art. Ideally, the dosage
of efflux pump inhibitor administered will be sufficient to reduce
efflux pump activity such that standard doses of drugs have a
therapeutic effect, e.g., result in a benefit to the subject, e.g.,
by inhibiting microbial growth. The phrase "therapeutic effect"
refers to an amelioration of symptoms or a prolongation of survival
in a subject. In a preferred embodiment, a therapeutic effect is an
elimination of a microbial infection.
[0078] In one embodiment, the subject is an avian subject, e.g., a
chicken or a turkey. In another embodiment, the subject is a
mammalian subject, e.g., a horse, sheep, pig, cow, dog, or cat. In
a preferred embodiment, the subject is a human subject.
[0079] In one embodiment, an infection in a subject is treated
prophylacticly. The term "prophylactic" treatment refers to
treating a subject who is not yet infected, but who is susceptible
to, or at risk of an infection. In one embodiment, the efflux pump
inhibitor is administered prior to exposure to an infectious agent.
In another embodiment, an efflux pump inhibitor is administered to
a subject prior to the exposure of the subject to a drug resistant
organism. The term "therapeutic" treatment refers to administering
a compound to a subject already suffering from an infection.
[0080] In one embodiment an efflux pump inhibitor and/or drug may
be administered in prodrug form, e.g., may be administered in a
form which is modified within the cell to produce the functional
form of the efflux pump inhibitor or fluoroquinolone.
[0081] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal for 50% of the population) and the ED50 (the does
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio between LD50/ED50. Compounds which
exhibit large therapeutic indices are preferred. The data obtained
from these cell culture assays and animal studies can be used in
formulating a range of dosage for use in human subjects. The dosage
of such compounds lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage may vary within this range depending upon the dosage
form employed and the route of administration utilized. For
example, in one embodiment the therapeutic serum concentration of
an efflux pump inhibitor is in the range of 0.1-100 .mu.g/ml.
VI. Microbes
[0082] Numerous different microbes are suitable for use in testing
for compounds that affect fluoroquinolone resistance or as sources
of materials for use in the instant assays or as targets for growth
inhibition. The term "microbe" includes any microorganism having a
an AcrAB-like efflux pump. Preferably unicellular microbes
including bacteria, fungi, or protozoa. In another embodiment,
microbes suitable for use in the invention are multicellular, e.g.,
parasites or fungi. In preferred embodiments, microbes are
pathogenic for humans, animals, or plants. As such, any of these
disclosed microbes may be used as intact cells or as sources of
materials for cell-free assays as described herein.
[0083] In preferred embodiments, microbes for use in the claimed
methods are bacteria, either Gram-negative or Gram-positive
bacteria. In a preferred embodiment, any bacteria that are shown to
become resistant to antibiotics, e.g., to display MDR are
appropriate for use in the claimed methods.
[0084] In preferred embodiments, microbes suitable for testing are
bacteria from the family Enterobacteriaceae. In more preferred
embodiments bacteria of a genus selected from the group consisting
of: Escherichia, Proteus, Salmonella, Klebsiella, Providencia,
Enterobacter, Burkholderia, Pseudomonas, Acinetobacter, Aeromonas,
Haemophilus, Yersinia, Neisseria, and Erwinia, Rhodopseudomonas, or
Burkholderia is used in the claimed assays.
[0085] In yet other embodiments, the microbes to be tested are
Gram-positive bacteria and are from a genus selected from the group
consisting of: Lactobacillus, Azorhizobium, Streptococcus,
Pediococcus, Photobacterium, Bacillus, Enterococcus,
Staphylococcus, Clostridium, Butyrivibrio, Sphingomonas,
Rhodococcus, or Streptomyces
[0086] In yet other embodiments, the microbes to be tested are acid
fast bacilli, e.g., from the genus Mycobacterium.
[0087] In still other embodiments, the microbes to be tested are,
e.g., selected from a genus selected from the group consisting of:
Methanobacterium, Sulfolobus, Archaeoglobu, Rhodobacter, or
Sinorhizobium.
[0088] In other embodiments, the microbes to be tested are fungi.
In a preferred embodiment the fungus is from the genus Mucor or
Candida, e.g., Mucor racemosus or Candida albicans.
[0089] In yet other embodiments, the microbes to be tested are
protozoa. In a preferred embodiment the microbe is a malaria or
cryptosporidium parasite.
[0090] In one embodiment, the microbe is resistant to one or more
drugs. In a preferred embodiment, the microbe is highly resistant
to one or more drugs. In one embodiment, the drug is an antibiotic.
In a preferred embodiment, the drug is a fluoroquinolone. In
another embodiment, the drug is a non-antibiotic. In another
embodiment, the drug is triclosan. In one embodiment, a microbe
comprises a mutation in a gene which is a target of the drug to
which the microbe is resistant, e.g., topoisomerase, gyrase, or
fabI gene. In another embodiment, a microbe comprises a mutation in
at least two of a topoisomerase, gyrase, or fabI gene.
VII. Test Compounds
[0091] Compounds for testing in the instant methods can be derived
from a variety of different sources and can be known or can be
novel. In one embodiment, libraries of compounds are tested in the
instant methods to identify AcrAB-like efflux pump blocking agents.
In another embodiment, known compounds are tested in the instant
methods to identify AcrAB-like efflux pump blocking agents. In a
preferred embodiment, compounds among the list of compounds
generally regarded as safe (GRAS) by the Environmental Protection
Agency are tested in the instant methods. In one embodiment, an
AcrAB-like efflux pump inhibitor is itself a substrate of the
pump.
[0092] A recent trend in medicinal chemistry includes the
production of mixtures of compounds, referred to as libraries.
While the use of libraries of peptides is well established in the
art, new techniques have been developed which have allowed the
production of mixtures of other compounds, such as benzodiazepines
(Bunin et al. 1992. J. Am. Chem. Soc. 114:10987; DeWitt et al.
1993. Proc. Natl. Acad. Sci. USA 90:6909) peptoids (Zuckermann.
1994. J. Med. Chem. 37:2678) oligocarbamates (Cho et al. 1993.
Science. 261:1303), and hydantoins (DeWitt et al. supra). Rebek et
al. have described an approach for the synthesis of molecular
libraries of small organic molecules with a diversity of 104-105
(Carell et al. 1994. Angew. Chem. Int. Ed. Engl. 33:2059; Carell et
al. Angew. Chem. Int. Ed. Engl. 1994. 33:2061).
[0093] The compounds for screening in the assays of the present
invention can be obtained using any of the numerous approaches in
combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or
solution phase libraries, synthetic library methods requiring
deconvolution, the `one-bead one-compound` library method, and
synthetic library methods using affinity chromatography selection.
The biological library approach is limited to peptide libraries,
while the other four approaches are applicable to peptide,
non-peptide oligomer or small molecule libraries of compounds (Lam,
K. S. Anticancer Drug Des. 1997. 12:145).
[0094] Exemplary compounds which can be screened for activity
include, but are not limited to, peptides, nucleic acids,
carbohydrates, small organic molecules (e.g., polyketides) (Cane et
al. 1998. Science 282:63), and natural product extract libraries.
In one embodiment, the test compound is a peptide or
peptidomimetic. In another, preferred embodiment, the compounds are
small, organic non-peptidic compounds.
[0095] Other exemplary methods for the synthesis of molecular
libraries can be found in the art, for example in: Erb et al. 1994.
Proc. Natl. Acad. Sci. USA 91:11422; Horwell et al. 1996
Immunopharmacology 33:68; and in Gallop et al. 1994. J. Med. Chem.
37:1233.
[0096] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra). Other types of peptide
libraries may also be expressed, see, for example, U.S. Pat. Nos.
5,270,181 and 5,292,646). In still another embodiment,
combinatorial polypeptides can be produced from a cDNA library.
VIII. Pharmaceutical Compositions
[0097] The invention provides pharmaceutically acceptable
compositions which include a therapeutically-effective amount or
dose of an efflux pump inhibitor and one or more pharmaceutically
acceptable carriers (additives) and/or diluents. A composition can
also include a second antimicrobial agent, e.g., an antimicrobial
compound, preferably an antibiotic, e.g., a fluoroquinolone.
[0098] As described in detail below, the pharmaceutical
compositions can be formulated for administration in solid or
liquid form, including those adapted for the following: (1) oral
administration, for example, drenches (aqueous or non-aqueous
solutions or suspensions), tablets, boluses, powders, granules,
pastes; (2) parenteral administration, for example, by
subcutaneous, intramuscular or intravenous injection as, for
example, a sterile solution or suspension; (3) topical application,
for example, as a cream, ointment or spray applied to the skin; (4)
intravaginally or intrarectally, for example, as a pessary, cream,
foam, or suppository; or (5) aerosol, for example, as an aqueous
aerosol, liposomal preparation or solid particles containing the
compound.
[0099] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting the antimicrobial agents or compounds of the invention
from one organ, or portion of the body, to another organ, or
portion of the body without affecting its biological effect. Each
carrier should be "acceptable" in the sense of being compatible
with the other ingredients of the composition and not injurious to
the subject. Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
compositions. Proper fluidity can be maintained, for example, by
the use of coating materials, such as lecithin, by the maintenance
of the required particle size in the case of dispersions, and by
the use of surfactants.
[0100] These compositions may also contain additional agents, such
as preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
[0101] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0102] Pharmaceutical compositions of the present invention may be
administered to epithelial surfaces of the body orally,
parenterally, topically, rectally, nasally, intravaginally,
intracisternally. They are of course given by forms suitable for
each administration route. For example, they are administered in
tablets or capsule form, by injection, inhalation, eye lotion,
ointment, etc., administration by injection, infusion or
inhalation; topical by lotion or ointment; and rectal or vaginal
suppositories.
[0103] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0104] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a sucrose
octasulfate and/or an antibacterial or a contraceptive agent, drug
or other material other than directly into the central nervous
system, such that it enters the subject's system and, thus, is
subject to metabolism and other like processes, for example,
subcutaneous administration.
[0105] In some methods, the compositions of the invention can be
topically administered to any epithelial surface. An "epithelial
surface" according to this invention is defined as an area of
tissue that covers external surfaces of a body, or which and lines
hollow structures including, but not limited to, cutaneous and
mucosal surfaces. Such epithelial surfaces include oral,
pharyngeal, esophageal, pulmonary, ocular, aural, nasal, buccal,
lingual, vaginal, cervical, genitourinary, alimentary, and
anorectal surfaces.
[0106] Compositions can be formulated in a variety of conventional
forms employed for topical administration. These include, for
example, semi-solid and liquid dosage forms, such as liquid
solutions or suspensions, suppositories, douches, enemas, gels,
creams, emulsions, lotions, slurries, powders, sprays, lipsticks,
foams, pastes, toothpastes, ointments, salves, balms, douches,
drops, troches, chewing gums, lozenges, mouthwashes, rinses.
[0107] Conventionally used carriers for topical applications
include pectin, gelatin and derivatives thereof, polylactic acid or
polyglycolic acid polymers or copolymers thereof, cellulose
derivatives such as methyl cellulose, carboxymethyl cellulose, or
oxidized cellulose, guar gum, acacia gum, karaya gum, tragacanth
gum, bentonite, agar, carbomer, bladderwrack, ceratonia, dextran
and derivatives thereof, ghatti gum, hectorite, ispaghula husk,
polyvinypyrrolidone, silica and derivatives thereof, xanthan gum,
kaolin, talc, starch and derivatives thereof, paraf fin, water,
vegetable and animal oils, polyethylene, polyethylene oxide,
polyethylene glycol, polypropylene glycol, glycerol, ethanol,
propanol, propylene glycol (glycols, alcohols), fixed oils, sodium,
potassium, aluminum, magnesium or calcium salts (such as chloride,
carbonate, bicarbonate, citrate, gluconate, lactate, acetate,
gluceptate or tartrate).
[0108] Such compositions can be particularly useful, for example,
for treatment or prevention of an unwanted cell, e.g., vaginal
Neisseria gonorrhea, or infections of the oral cavity, including
cold sores, infections of eye, the skin, or the lower intestinal
tract. Standard composition strategies for topical agents can be
applied to the antimicrobial compounds, or pharmaceutically
acceptable salts thereof in order to enhance the persistence and
residence time of the drug, and to improve the prophylactic
efficacy achieved.
[0109] For topical application to be used in the lower intestinal
tract or vaginally, a rectal suppository, a suitable enema, a gel,
an ointment, a solution, a suspension or an insert can be used.
Topical transdermal patches may also be used. Transdermal patches
have the added advantage of providing controlled delivery of the
compositions of the invention to the body. Such dosage forms can be
made by dissolving or dispersing the agent in the proper
medium.
[0110] Compositions of the invention can be administered in the
form of suppositories for rectal or vaginal administration. These
can be prepared by mixing the agent with a suitable non-irritating
carrier which is solid at room temperature but liquid at rectal
temperature and therefore will melt in the rectum or vagina to
release the drug. Such materials include cocoa butter, beeswax,
polyethylene glycols, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active agent.
[0111] Compositions which are suitable for vaginal administration
also include pessaries, tampons, creams, gels, pastes, foams,
films, or spray compositions containing such carriers as are known
in the art to be appropriate. The carrier employed in the sucrose
octasulfate/contraceptive agent should be compatible with vaginal
administration and/or coating of contraceptive devices.
Combinations can be in solid, semi-solid and liquid dosage forms,
such as diaphragm, jelly, douches, foams, films, ointments, creams,
balms, gels, salves, pastes, slurries, vaginal suppositories,
sexual lubricants, and coatings for devices, such as condoms,
contraceptive sponges, cervical caps and diaphragms.
[0112] For ophthalmic applications, the pharmaceutical compositions
can be formulated as micronized suspensions in isotonic, pH
adjusted sterile saline, or, preferably, as solutions in isotonic,
pH adjusted sterile saline, either with or without a preservative
such as benzylalkonium chloride. Alternatively, for ophthalmic
uses, the compositions can be formulated in an ointment such as
petrolium. Exemplary ophthalmic compositions include eye ointments,
powders, solutions and the like.
[0113] Powders and sprays can contain, in addition to sucrose
octasulfate and/or antibiotic or contraceptive agent(s), carriers
such as lactose, talc, silicic acid, aluminum hydroxide, calcium
silicates and polyamide powder, or mixtures of these substances.
Sprays can additionally contain customary propellants, such as
chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,
such as butane and propane.
[0114] Ordinarily, an aqueous aerosol is made by formulating an
aqueous solution or suspension of the agent together with
conventional pharmaceutically acceptable carriers and stabilizers.
The carriers and stabilizers vary with the requirements of the
particular compound, but typically include nonionic surfactants
(Tweens, Pluronics, or polyethylene glycol), innocuous proteins
like serum albumin, sorbitan esters, oleic acid, lecithin, amino
acids such as glycine, buffers, salts, sugars or sugar alcohols.
Aerosols generally are prepared from isotonic solutions.
[0115] Compositions of the invention can also be orally
administered in any orally-acceptable dosage form including, but
not limited to, capsules, cachets, pills, tablets, lozenges (using
a flavored basis, usually sucrose and acacia or tragacanth),
powders, granules, or as a solution or a suspension in an aqueous
or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid
emulsion, or as an elixir or syrup, or as pastilles (using an inert
base, such as gelatin and glycerin, or sucrose and acacia) and/or
as mouth washes and the like, each containing a predetermined
amount of sucrose octasulfate and/or antibiotic or contraceptive
agent(s) as an active ingredient. A compound may also be
administered as a bolus, electuary or paste. In the case of tablets
for oral use, carriers which are commonly used include lactose and
corn starch. Lubricating agents, such as magnesium stearate, are
also typically added. For oral administration in a capsule form,
useful diluents include lactose and dried corn starch. When aqueous
suspensions are required for oral use, the active ingredient is
combined with emulsifying and suspending agents. If desired,
certain sweetening, flavoring or coloring agents may also be
added.
[0116] Tablets, and other solid dosage forms, such as dragees,
capsules, pills and granules, may be scored or prepared with
coatings and shells, such as enteric coatings and other coatings
well known in the pharmaceutical-formulating art. They may also be
formulated so as to provide slow or controlled release of the
active ingredient therein using, for example, hydroxypropylmethyl
cellulose in varying proportions to provide the desired release
profile, other polymer matrices, liposomes and/or microspheres.
They may be sterilized by, for example, filtration through a
bacteria-retaining filter, or by incorporating sterilizing agents
in the form of sterile solid compositions which can be dissolved in
sterile water, or some other sterile injectable medium immediately
before use. These compositions may also optionally contain
opacifying agents and may be of a composition that they release the
active ingredient(s) only, or preferentially, in a certain portion
of the gastrointestinal tract, optionally, in a delayed manner.
Examples of embedding compositions which can be used include
polymeric substances and waxes. The active ingredient can also be
in micro-encapsulated form, if appropriate, with one or more of the
above-described excipients.
[0117] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
ingredient, the liquid dosage forms may contain inert diluents
commonly used in the art, such as, for example, water or other
solvents, solubilizing agents and emulsifiers, such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
oils (in particular, cottonseed, groundnut, corn, germ, olive,
castor and sesame oils), glycerol, tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and
mixtures thereof.
[0118] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0119] Suspensions, in addition to the antimicrobial agent(s) may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0120] Sterile injectable forms of the compositions of this
invention can be aqueous or oleaginous suspensions. These
suspensions may be formulated according to techniques known in the
art using suitable dispersing or wetting agents and suspending
agents. Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0121] The sterile injectable preparation may also be a sterile
injectable solution or suspension in a nontoxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed including
synthetic mono- or di-glycerides. Fatty acids, such as oleic acid
and its glyceride derivatives are useful in the preparation of
injectables, as are natural pharmaceutically-acceptable oils, such
as olive oil or castor oil, especially in their polyoxyethylated
versions. These oil solutions or suspensions may also contain a
long-chain alcohol diluent or dispersant.
[0122] In the case of AcrAB efflux pump inhibitors which are
antisense nucleic acid molecules, the optimal course of
administration of the oligomers may vary depending upon the desired
result or on the subject to be treated. As used in this context
"administration" refers to contacting cells with oligomers. The
dosage of antisense molecule may be adjusted to optimally reduce
expression of a protein translated from a target mRNA, e.g., as
measured by a readout of RNA stability or by a therapeutic
response, without undue experimentation. For example, expression of
the protein encoded by the nucleic acid target can be measured to
determine whether or dosage regimen needs to be adjusted
accordingly. In addition, an increase or decrease in RNA and/or
protein levels in a cell or produced by a cell can be measured
using any art recognized technique. By determining whether
transcription has been decreased, the effectiveness of the
antisense molecule in inducing the cleavage of the target RNA can
be determined.
[0123] As used herein, "pharmaceutically acceptable carrier"
includes appropriate solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, it can be used in the therapeutic compositions.
Supplementary active ingredients can also be incorporated into the
compositions.
[0124] Antisense molecule may be incorporated into liposomes or
liposomes modified with polyethylene glycol or admixed with
cationic lipids for parenteral administration. Incorporation of
additional substances into the liposome, for example, antibodies
reactive against membrane proteins found on specific target
microbes, can help target the antisense molecule to specific cell
types.
[0125] Moreover, the present invention provides for administering
the subject oligomers with an osmotic pump providing continuous
infusion of such antisense molecules, for example, as described in
Rataiczak et al. (1992 Proc. Natl. Acad. Sci. USA 89:11823-11827).
Such osmotic pumps are commercially available, e.g., from Alzet
Inc. (Palo Alto, Calif.). Topical administration and parenteral
administration in a cationic lipid carrier are preferred.
[0126] With respect to in vivo applications, the formulations of
the present invention can be administered to a patient in a variety
of forms adapted to the chosen route of administration, namely,
parenterally, orally, or intraperitoneally. Parenteral
administration, which is preferred, includes administration by the
following routes: intravenous; intramuscular; interstitially;
intraarterially; subcutaneous; intra ocular; intrasynovial; trans
epithelial, including transdermal; pulmonary via inhalation;
ophthalmic; sublingual and buccal; topically, including ophthalmic;
dermal; ocular; rectal; and nasal inhalation via insufflation.
Intravenous administration is preferred among the routes of
parenteral administration.
[0127] Pharmaceutical preparations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
or water-dispersible form. In addition, suspensions of the active
compounds as appropriate oily injection suspensions may be
administered. Suitable lipophilic solvents or vehicles include
fatty oils, for example, sesame oil, or synthetic fatty acid
esters, for example, ethyl oleate or triglycerides. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension include, for example, sodium
carboxymethyl cellulose, sorbitol, and/or dextran, optionally, the
suspension may also contain stabilizers.
[0128] Drug delivery vehicles can be chosen e.g., for in vitro, for
systemic, or for topical administration. These vehicles can be
designed to serve as a slow release reservoir or to deliver their
contents directly to the target cell. An advantage of using some
direct delivery drug vehicles is that multiple molecules are
delivered per uptake. Such vehicles have been shown to increase the
circulation half-life of drugs that would otherwise be rapidly
cleared from the blood stream. Some examples of such specialized
drug delivery vehicles which fall into this category are liposomes,
hydrogels, cyclodextrins, biodegradable nanocapsules, and
bioadhesive microspheres.
[0129] The described antisense molecules may be administered
systemically to a subject. Systemic absorption refers to the entry
of drugs into the blood stream followed by distribution throughout
the entire body. Administration routes which lead to systemic
absorption include: intravenous, subcutaneous, intraperitoneal, and
intranasal. Each of these administration routes delivers the
antisense molecule to accessible diseased cells. Following
subcutaneous administration, the therapeutic agent drains into
local lymph nodes and proceeds through the lymphatic network into
the circulation. The rate of entry into the circulation has been
shown to be a function of molecular weight or size. The use of a
liposome or other drug carrier localizes the antisense molecule at
the lymph node. The antisense molecule can be modified to diffuse
into the cell, or the liposome can directly participate in the
delivery of either the unmodified or modified oligomer into the
cell.
[0130] For prophylactic applications, the pharmaceutical
composition of the invention can be applied prior to physical
contact with a microbe. The timing of application prior to physical
contact can be optimized to maximize the prophylactic effectiveness
of the compound. The timing of application will vary depending on
the mode of administration, the epithelial surface to which it is
applied, the surface area, doses, the stability and effectiveness
of composition under the pH of the epithelial surface, the
frequency of application, e.g., single application or multiple
applications. Preferably, the timing of application can be
determined such that a single application of composition is
sufficient. One skilled in the art will be able to determine the
most appropriate time interval required to maximize prophylactic
effectiveness of the compound.
[0131] One of ordinary skill in the art can determine and prescribe
the effective amount of the pharmaceutical composition required.
For example, one could start doses at levels lower than that
required in order to achieve the desired therapeutic effect and
gradually increase the dosage until the desired effect is achieved.
In general, a suitable daily dose of a composition of the invention
will be that amount of the composition which is the lowest dose
effective to produce a therapeutic effect. Such an effective dose
will generally depend upon the factors described above. It is
preferred that administration be intravenous, intracoronary,
intramuscular, intraperitoneal, or subcutaneous.
[0132] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, genetics, microbiology, recombinant
DNA, and immunology, which are within the skill of the art. Such
techniques are explained fully in the literature. See, for example,
Genetics; Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by
Sambrook, J. et al. (Cold Spring Harbor Laboratory Press (1989));
Short Protocols in Molecular Biology, 3rd Ed., ed. by Ausubel, F.
et al. (Wiley, NY (1995)); DNA Cloning, Volumes I and II (D. N.
Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.
(1984)); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. (1984)); the
treatise, Methods In Enzymology (Academic Press, Inc., N.Y);
Immunochemical Methods In Cell And Molecular Biology (Mayer and
Walker, eds., Academic Press, London (1987)); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds. (1986)); and Miller, J. Experiments in Molecular
Genetics (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
(1972)).
[0133] The contents of all references, pending patent applications
and published patents, cited throughout this application are hereby
expressly incorporated by reference.
[0134] The invention is further illustrated by the following
examples, which should not be construed as further limiting.
EXAMPLES
Example 1
Preventing Efflux Via An AcrAB Pump Renders Highly Resistant
Microbes Comprising Chromosomal Mutations in Drug Target Genes
Sensitive to Drugs
Ineffectiveness of Gyrase Mutations in Escherichia coli in the
Absence of the AcrAB Multidrug Efflux Pump.
[0135] Fluoroquinolones [FQs] inhibit growth of wild-type
Escherichia coli at very low concentrations. While FQ efflux is
present in wild-type E. coli cells, its importance is minimal
except when enhanced during the development of clinical resistance.
To understand the role of the AcrAB efflux pump, which is under
control of the mar and sox regulons, on the level of susceptibility
and clinical resistance to FQs in E. coli, two series of E. coli
K-12 cells (derived from wild type AG100 and from the isogenic Mar
mutant AG102) with FQ resistance were generated by amplification on
ofloxacin [ofx]-containing plates in-vitro and mutations conferring
resistance were characterized. P1-transduction was used to knock
out AcrAB. Energy-dependent uptake of radiolabelled ciprofloxacin
[cfx] into whole cells was measured in the presence and absence of
AcrAB, and ofx MICs were determined by broth microdilution. In
AG100, ofx resistance could be enhanced from 0.03 mg/l to 4 mg/l
(128-fold) in four steps which included (in the order of
occurrence): a gyrA mutation, a mar mutation, an undefined
mutation, and a 2.sup.nd gyrA mutation. AG102, starting out as a
Mar mutant, was amplified in three steps from 0.125 mg/l to 8 mg/l
(64-fold), which included one gyrA mutation and two as yet
undefined mutations. Knock-out of AcrAB reduced the ofx MICs by
factors of 4 through 128, producing a dramatic decrease in
resistance mediated by topoisomerase mutations. Ofx MICs.+-.AcrAB
were as follows (in mg/1): AG100 series: 0.03/<0.015, 0.25/0.06,
1.0/0.06, 2.0/0.06, 4.0/0.125; AG102 series: 0.125/<0.015,
1.0/0.06, 4.0/0.06, 8.0/0.06. Active efflux of cfx was seen in the
presence of the AcrAB efflux pump. Drug accumulation in energized,
AcrAB-deleted mutants, however, was equivalent to that in
de-energized cells, i.e., active efflux was completely abolished by
deletion of AcrAB. Thus, the AcrAB multidrug efflux pump is the
major, if not only, efflux pump in E. coli which controls the
intracellular concentration of cfx. In its absence, high
intracellular drug concentrations render topoisomerase mutations
relatively ineffective in achieving clinical FQ resistance in E.
coli.
Deletion Of The AcrAB Efflux Pump Greatly Reduces Fluoroquinolone
Resistance In Gyrase Mutants Of Escherichia coli
[0136] Fluoroquinolone-resistant mutants were selected from a
wild-type Escherichia coli K12 strain and its Mar mutant by
stepwise exposure to increasing levels of ofloxacin on solid
medium. Analysis of mutational steps by Northern (RNA) blot
analysis, sequencing and accumulation studies with radiolabelled
ciprofloxacin showed that mutations in the target gene gyrA, the
regulatory gene marR and additional, as yet unidentified genes
probably affecting AcrAB-mediated efflux of ciprofloxacin all
contributed to fluoroquinolone resistance. Inactivation of the
acrAB locus made all strains hypersusceptible to fluoroquinolones
and certain other unrelated drugs. These studies indicate that
wild-type function of the AcrAB efflux pump is required for
expression of clinical fluoroquinolone resistance mediated by
topoisomerase mutations. Fluoroquinolone (FQ) resistance in
Escherichia coli can be caused by mutations in the target proteins
of the drugs, DNA gyrase (Hooper, D. C. et al., 1987. The American
Journal of Medicine 82:12-20; Piddock, L. J. V. 1995. Drugs
49:29-35) and topoisomerase IV (Heisig, P. 1996. Antimicrob. Agents
Chemother. 40:879-885). Mutations affecting regulatory genes such
as marA (Cohen, S. P., et al. 1993. J. Bacteriol. 175: 1484-1492,
Cohen, S. P., et al. 1989. Antimicrob. Agents Chemother.
33:1318-1325) or soxS (Amabile-Cuevas, C. F. and B. Demple. 1991.
Nucleic Acids Research 19:4479-4484) also lead to resistance. The
latter genes regulate intracellular drug concentrations, either by
decreased uptake and/or increased efflux of the drug (Alekshun, M.
N. and S. B. Levy. 1997. Agents Chemother. 41: 2067-2075). In E.
coli, overexpression of MarA causes decreased expression of the
OmpF porin (Cohen, S. P., et al. 1988. J. Bacteriol. 170:5416-5422)
and increased AcrAB expression (Okusu, H., et al. 1996. J.
Bacteriol. 178:306-308), thereby conferring resistance to a large
number of antimicrobials (Alekshun, M. N. and S. B. Levy. 1997.
Antimicrob. Agents Chemother. 41: 2067-2075)). The sequence of
events leading to high-level, clinically significant FQ resistance
is still poorly understood. Studies on clinical FQ-resistant
strains are of limited help because one usually cannot isolate the
parental strain. Studies with mutants selected in vitro have been
limited to descriptions of phenotypic differences (MICs, outer
membrane profiles, accumulation of fluoroquinolones) or focussed on
mutations in the regions of gyrA and/or parC which determine
quinolone resistance (QRDR) (Heisig, P. 1996. Antimicrob. Agents
Chemother. 40:879-885; Piddock, L. J. V., et al. 1991. J.
Antimicrob. Chemother. 28:185-198; Tenney, J. H., et al. 1983.
Antimicrob. Agents Chemother. 23:188-189; Watanabe, M., et al.
1990. Antimicrob. Agents Chemother. 34:173-175). In this in vitro
study the role of the AcrAB efflux pump was studied in FQ
resistance mediated by topoisomerase mutations acquired during
step-wise selection on ofloxacin. The AcrAB efflux pump was found
to be critical to the FQ resistance level.
The following materials and methods were used in this example:
[0137] Antibiotics, chemicals and media. Ofloxacin (OFL) was kindly
donated by Hoechst, Frankfurt, Germany. Radiolabelled
[.sup.14C-]ciprofloxacin was a generous gift of the Bayer AG,
Leverkusen, Germany. Carbonyl cyanide m-chlorophenylhydrazone
(CCCP) was purchased from Sigma Chemical Co., St. Louis, Mo., and
organic solvents from Aldrich, Milwaukee, Wis. Strains were grown
in LB broth (10 g of tryptone, 5 g of yeast extract and 10 g of
NaCl per liter) unless otherwise noted.
[0138] Bacterial strains and plasmids. All strains were derivatives
of plasmid-free E. coli K-12 strain AG100 (George, A. M. and S. B.
Levy. 1983. J. Bacteriol. 155:531-540) and its Mar mutant AG112
which was selected on tetracycline in two steps (this study).
Wild-type E. coli GC4468, its derived Sox mutant JTG1078 (soxR105;
(Greenberg, J. T., et al. 1991. J. Bact 173:4433-4439)) and plasmid
pSXS bearing the soxS gene on a 432 bp-fragment (Amabile-Cuevas, C.
F. and B. Demple. 1991. Nucleic Acids Research 19:4479-4484).
Strain JZM120 was as described (Ma, D., et al. 1995. Mol Microbiol.
16:45-55)
[0139] Selection of fluoroquinolone-resistant mutants. 1-AG100,
2.degree.-AG100, 3*-AG100, 4*-AG100 and 1'-AG112, 2'-AG112,
3'-AG112 were sequential step mutants derived from AG100 and AG112,
respectively, on solid media. For each step, about 10.sup.11 cells
of an overnight culture were plated on several LB agar plates
supplemented with increasing concentrations of OFL. Single colonies
were further purified on OFL-supplemented agar plates. Mutant
2.degree.-AG100 came out of one series, while mutants 3*- and
4*-AG100 came from a second series.
[0140] Susceptibility testing. MICs of selected antimicrobial
agents were determined by a standard broth microdilution procedure
with cation-adjusted Mueller-Hinton broth (Becton Dickinson,
Cockeysville, Md.) and an inoculum of 5.times.10.sup.5 CFU/ml
according to NCCLS performance and interpretive guidelines (NCCLS.
1997. Methods for dilution antimicrobial susceptibility. Tests for
bacteria that grow aerobically--Fourth Edition; Approved Standard.
NCCLS document M7-A4 (ISBN 1-56238-309-4). NCCLS, 940 West Valley
Road, Suite 1400, Wayne, Pa. 19087). Antimicrobial agents on the
commercially available microtiter plates (Merlin Diagnostics GmbH,
Bornheim, Germany) included the FQs ciprofloxacin (CIP), enoxacin,
fleroxacin, nor-flo-xacin, ofloxacin (OFL), pefloxacin,
sparfloxacin (SPX), and trovafloxacin (TVA). They also included
tetracycline (TET), chloramphenicol (CML), trimethoprim (TMP),
cefoxitin (CFOX), cefaclor, cefixim and loracarbef. MICs of bile
salts were determined by a broth macrodilution procedure: sodium
cholate and sodium deoxycholate were serially diluted twofold in LB
broth and tubes inoculated at 5.times.10.sup.5 CFU/ml. MICs of both
antibiotics and bile salts were determined twice in independent
experiments with reproducible results.
[0141] P1 transduction. AcrAB-deleted strains were constructed by
P1 transduction (Provence, D. L. and R. Curtiss III. 1994. In: P.
Gerhardt, R. G. E. Murray, W. A. Wood, and N. R. Krieg. (ed.),
Methods for General and Molecular Bacteriology. American Society
for Microbiology, Washington. pp. 317-347) of acrAB::Tn903kan.sup.r
from strain JZM120 (Ma, D., et al. 1995. Mol Microbiol. 16:45-55)
into AG100, AG112 and all derived FQ-resistant mutants. The
AcrAB-deleted strains were designated AG100AK, 1- through
4*-AG100AK, AG112AK and 1'- through 3'-AG112AK. Two independent
transductants were saved for each recipient. Deletion of AcrAB was
confirmed by the absence of intact target DNA in a PCR assay and by
greatly increased susceptibility to bile salts as previously
reported (Thanassi, D. G., et al. 1997. J. Bacteriol.
179:2512-2518).
[0142] DNA sequencing. The QRDRs of gyrA (nucleotides 123 to 366)
or parC (nucleotides 145 to 492) in the fluoroquinolone-resistant
mutants were amplified by PCR and purified by use of Qia-quick spin
columns (Qia-gen, Hilden, Germany), as previously described
(Conrad, S., M. et al. 1996. J. Antimicrob. Chemother. 38:443-455,
Conrad, S., et al. 1996. Program and Abstracts of the 36th
Interscience Conference of Anti-microbial Agents and Chemotherapy,
New Orleans. Abstract C9:35). marOR was amplified from bp 1311 to
1858 with primer pair ORAB2 and RK3 as described earlier
(Oethinger, M., et al. 1998. Agents Chemother. 42:2089-2094).
Direct cycle sequencing was performed in an automatic 373A DNA
Sequencer (Applied Biosystems).
[0143] RNA extraction and Northern blot analysis. Northern blot
analysis was performed as previously described in detail
(Oethinger, M., et al 1998. Antimicrob. Agents Chemother.
42:2089-2094). In brief, RNA was harvested by a cesium chloride
method from midlogarithmic phase cultures grown at 30.degree. C.
For assessment of the state of the marRAB operon or the soxRS
operon, respectively, cultures were split and half the cultures
induced with 5 mM sodium salicylate (marRAB operon) or 1.3 mM
paraquat (soxRS operon), respectively. The level of transcription
from both operons was assessed by hybridization of radiolabelled
DNA probes (marA or soxS) to the membrane-bound RNA (20
.mu.g/lane), exposure on a PhosphoImager screen, and visualization
with ImageQuant Software (Molecular Dynamics, Sunnyvale, Calif.),
as described recently (Oethinger, M., et al. 1998. Antimicrob.
Agents Chemother. 42:2089-2094).
[0144] Accumulation of [.sup.14C-]ciprofloxacin in whole cells.
Cultures were grown to logarithmic phase in LB broth at 30.degree.
C., washed in 50 mM potassium phosphate/0.2% glucose (pH 7.4), and
resuspended in the same buffer to OD.sub.600=5-7.
[.sup.14C-]ciprofloxa-cin (specific activity: 59 mCi/mmol) was
added to 10 .mu.M. Accumulation was measured at equilibrium after 5
and 15 min by dilution of 50 .mu.l of cell-labeling suspension into
5 ml of 100 mM LiCl/50 mM KPO.sub.4 (pH 7.4), collection of cells
immediately on Gelman metricel mixed-cellulose ester membrane
filters (pore size, 0.45 .mu.m; Gelman Sciences Inc., Ann Arbor,
Mich.), and washing with 5 ml of the same buffer. Filters were
dried, and radioactivity was assayed with a liquid scintillation
counter, using Betafluor (National Diagnostics, Somerville, N.J.).
Counting efficiency was 90%. Binding of radiolabel to filters in
the absence of cells was subtracted. For conversion purposes, 10
.mu.M CIP=3.15 .mu.g/ml. When used, carbonyl cyanide
m-chlorophenyl-hydrazone (CCCP), which destroys the proton motive
force, was added to a final concentration of 200 .mu.M, and
accumulation of ciprofloxacin was assayed 5 and 15 min thereafter.
The assay was designed in a way to investigate up to five strains
in one experiment and to include, in addition, AG112 as a control
strain. Results were calculated as accumulation of CIP in picomoles
per OD.sub.600 unit, where 1 OD.sub.600 unit represented the number
of cells in 1 ml when the OD.sub.600 was equal to 1 (approximately
10.sup.9 E. coli cells, about 0.3 mg of protein). The ratio of CIP
accumulation of energized cells divided by that of de-energized
(CCCP-treated) cells was used as an indirect measure of active
efflux (Levy, S. B. 1992. Antimicrob. Agents Chemother.
36:695-703). For these calculations, results obtained before (5 and
15 min) and after adding CCCP (25 min and 35 min) were averaged and
ratios expressed as % of de-energized (i.e. maximum)
accumulation.
[0145] The mutation frequencies of the different step mutants
ranged between 8.times.10-.sup.8 and 10-.sup.10 which agrees well
with previous data (Heisig, P. 1996. Antimicrob. Agents Chemother.
40:879-885, Piddock, L. J. V., et al. 1991. J. Antimicrob.
Chemother. 28:185-198, Watanabe, M., et al. 1990. Antimicrob.
Agents Chemother. 34:173-175). None of the mutants was defective in
growth. The first mutation step increased resistance to OFL by
8-fold in both AG100 and its Mar mutant AG112 (Table 1). Subsequent
increases were 2- to 4-fold in all steps (Table 1 and Table 2)
yielding the highest MIC.sub.OFL of 8 .mu.g/ml in 3'-AG112. MICs of
all FQs increased in parallel, with the order of MICs being:
OFL>CIP>TVA=SPX (Table 2).
[0146] Identification of chromosomal mutations in structural and
regulatory genes, and susceptibilities to unrelated antibiotics.
Sequencing of the QRDRs of gyrA revealed that an identical point
mutation at codon 87 (substitution of glycine for aspartate)
occurred during the first mutation step in both AG100- and
AG112-derived mutants (Table 1). These mutations led to an increase
of MICs to FQs without an additional multiply resistance (Mar)
phenotype. There are several reports about a gyrA mutation being
the first "visible" mutation in the chain of events to higher FQ
resistance during stepwise in vitro mutagenesis (Heisig, P. 1996.
Antimicrob. Agents Chemother. 40:879-885, Kern, W. V., et al. 1997.
Program and Abstracts of the 37th Interscience Conference of
Antimicrobial Agents and Chemotherapy, Toronto (Abstract), Piddock,
L. J. V., et al. 1991. J. Antimicrob. Chemother. 28:185-198). The
concordance between AG100 and AG112 in the position of the first
gyrA mutation may be coincidental.
[0147] The second step mutation in the AG100 background was a Mar
mutation, as shown by overexpression of marRAB by Northern blot
analysis of 2.degree.-AG100. Such overexpression was found in three
independently selected second step mutants of AG100. Constitutive
overexpression of marA was associated with increased resistance to
TET, CML and CFOX (Table 2). Retrospective sequencing of marOR
showed that third step mutant 3*-AG100 had not been derived from
2.degree.-AG100 (Table 1). Despite many efforts, the putative
second step Mar mutant 2*-AG100 with a 1643G.fwdarw.T transition in
marOR could not be retrieved and further studied.
[0148] As expected (George, A. M. and S. B. Levy. 1983. J.
Bacteriol. 155:531-540), marRAB was derepressed in Mar mutant AG112
and all subsequently derived mutants. Sequencing of marOR of the
parental strain AG112 identified a 5 base pair deletion after the
codon for amino acid 12, resulting in deletion of one amino acid
and change of the complete protein sequence thereafter (Table 1).
These data indicate that a single mutation in the gyrA gene confers
a somewhat higher resistance than overexpression of marA by itself
(MIC.sub.OFL=0.25 .mu.g/ml (8-fold) vs. 0.125 .mu.g/ml (4-fold);
1-AG100 vs. AG112, Table 1) and that the two mutations are
multiplicative (MIC.sub.OFL=1 .mu.g/ml (32-fold); 2.degree.-AG100
and 1'-AG112, Table 1). During further steps to higher FQ
resistance only one mutant, 4*-AG100, acquired a second mutation in
gyrA at codon 83, substituting leucine for serine (Table 1). No
additional mutations in gyrA, parC or marOR could be identified in
any of the more resistant mutants, nor did they constitutively
overexpress soxS at any step. Mutant 2'-AG112, derived from
gyrA/Mar double mutant 1'-AG112, dis-played increased resistance to
multiple drugs (Table 2) with no further mutation in marOR. The
additional mutation possibly leads to upregulation of acrAB. In
contrast, next step mutant 3'-AG112 had increased resistance to
only FQs and CFOX (Table 2). The molecular basis for this
resistance is also unknown.
[0149] Accumulation of CIP into whole energized cells reached a
plateau by 5 min and averaged 103 pmol CIP/ODU.sub.600 in AG100.
When the proton motive force was dissipated by adding 200 .mu.M
CCCP, accumulation of [.sup.14C-]ciprofloxacin doubled (201 pmol
CEP/ODU.sub.600). These findings confirmed earlier studies with
norfloxacin (Cohen, S. P., et al. 1988. Antimicrob. Agents
Chemother. 32:1187-1191) that FQ-susceptible E. coli cells use
energy to reduce FQ accumulation, i.e. show active efflux. This
phenomenon was also observed for Proteus vulgaris using ofloxacin
(Ishii, H., et al. 1991. J. Antimicrob. Chemother. 28:827-836). In
comparison, accumulation in AG112 was 54% of wild-type, averaging
56 pmol CIP/ODU.sub.600, and increased to 226 pmol CIP/ODU.sub.600
after CCCP. The rapid accumulation of CIP or norfloxacin (Cohen, S.
P., et al. 1988. Antimicrob. Agents Chemother. 32:1187-1191) to the
level of the AG100 parental strains upon deenergization of cells
rules out down-regulation of outer membrane porins as the major
mechanism of reduced drug accumulation in Mar mutants. The amount
of CIP accumulated by energized first step mutants 1-AG100 and
1'-AG112 and the increase in drug uptake following deenergization
was virtually identical to that of parental strains AG100 and
AG112, respectively. In second step Mar mutant 2.degree.-AG100,
accumulation decreased to 65 pmol CIP/ODU.sub.600 (63% of
wild-type; FIG. 1). Independently isolated mutants 3*- and 4*-AG100
showed even greater reduced accumulation (16 pmol CIP/ODU.sub.600=,
16% of wild-type; and 20 pmol CIP/ODU.sub.600, 19% of wild-type,
respectively). Similarly, mutants 2'-AG112 and 3'-AG112 accumulated
considerably less CIP than Mar mutants AG112 and 1'-AG112 (26 pmol
CIP/ODU.sub.600 for both strains vs. 56 pmol CIP/ODU.sub.600 and 57
pmol CIP/ODU.sub.600, respectively, or 25% vs. 54% and 55% of
wild-type; FIG. 1). Due to more efficient efflux of the drug, the
intracellular concentration of CIP in 3*-/4*-AG100 and 2'-/3'-AG112
at high external drug levels is likely similar to that of the
parental strains at lower levels, so that the mutants can survive a
higher extracellular FQ concentration. Whatever mechanism was
underlying, it also increased the cells' resistance to TET, CML and
CFOX.
[0150] Effects of deletion of acrAB. Upon deletion of the AcrAB
multidrug efflux pump, active efflux of CIP was completely aborted
in all strains; energized AG100 cells bearing the .DELTA.acrAB
deletion accumulated CIP to more than twice the level seen for
energized acrAB.sup.+ cells (FIG. 1). No difference in CIP uptake
was noted between any strains bearing .DELTA.acrAB irrespective of
their mutations in marRAB or gyrA (FIG. 1). Although all
.DELTA.acrAB strains became profoundly hyper-susceptible to all
FQs, mutants with newly acquired mutations in gyrA (Table 1)
probably still retained the expected fold FQ resistance (Table 2).
This resistance, however, was well below clinical significance.
GyrA double mutant 4*-AG100.DELTA.acrAB, for instance, displayed a
MIC.sub.OFL of only 0.125 .mu.g/ml. Interestingly, the differences
in MICs for different FQs were no longer observed in .DELTA.acrAB
strains, e.g. in 2.degree.-AG100 .DELTA.acrAB.sup.+,
MIC.sub.OFL=MIC.sub.CIP=MIC.sub.TVA=0.06 .mu.g/ml, while in the
acrAB.sup.+ strain 2.degree.-AG100 MIC.sub.OFL=1 gg/ml,
MIC.sub.CIP=0.5 .mu.g/ml, MIC.sub.TVA=0.25 .mu.g/ml (Table 2). For
SPX MICs of all .DELTA.acrAB mutants were below detection
(MIC.sub.SPX .DELTA.0.015 .mu.g/ml). This may reflect a much higher
proportion of drug being effluxed by the AcrAB pump in energized
cells. Deletion of acrAB also eliminated the multidrug resistance
conferred by overexpression of marA, thus underlining previous
results that the AcrAB multidrug efflux pump plays a major role in
the antibiotic resistance phenotype of Mar mutants (Okusu, H., D.
Ma, and H. Nikaido. 1996. J. Bacteriol. 178:306-308). Effects of
additional, as yet unknown mutations were also completely abolished
by deletion of acrAB (Table 2). Thus, the effect of one or more
additional mutation(s) on drug resistance may act via acrAB.
[0151] Concluding Remarks. An earlier report on selection of
norfloxacin-resistant E. coli noted no change of MICs of seven
unrelated antibiotics during in vitro amplification of FQ-resistant
mutants (Tenney, J. H., et al. 1983. Antimicrob. Agents Chemother.
23:188-189). Another study showed that only two out of ten second
step mutants derived from a gyrA mutant displayed a
multiply-resistant phenotype (Piddock, L. J. V., et al. 1991. J.
Antimicrob. Chemother. 28:185-198).
[0152] The results of this study contrast with those previously
reported: the stepwise sequence of mutational events during in
vitro amplification of FQ resistance in three independent series
involved an initial gyrA mutation followed by a mutation causing
overexpression of the mar locus. A marOR mutation was also seen
during the second mutation step during selection of FQ-resistant E.
coli in vitro by others (Bagel, S., et al. 1999. Antimicrob. Agents
Chemother. 43:868-875).
[0153] In recent experiments, identical in vitro experimental
conditions were applied to select mutants of two clinical E. coli
isolates (Kern, W. V., et al. 1997. Program and Abstracts of the
37th Interscience Conference of Antimicrobial Agents and
Chemotherapy, Toronto (Abstract)). Since the second step mutant was
a Mar mutant in one strain and a Sox mutant in the other (Kern, W.
V., et al. 1997. Program and Abstracts of the 37th Interscience
Conference of Antimicrobial Agents and Chemotherapy, Toronto
(Abstract)), thus, a mutation in a regulatory gene occurs
frequently. However, studies on FQ-resistant E. coli of clinical
origin have shown that the proportion of constitutive Mar or Sox
mutants is only between 10% and 15% (Oethinger, M., et al. 1998.
Antimicrob. Agents Chemother. 42:2089-2094), a lower frequency than
seen in the present in vitro experiments.
[0154] In view of the high number of pumps in E. coli (Nikaido, H.
1996. J. Bacteriol. 178:5853-5859, Paulsen, I. T., et al. 1996.
Microbiol. Rev. 60:575-608) it is surprising that none of the other
pumps actively effluxes CIP in the absence of AcrAB pump. Thus the
AcrAB multidrug efflux pump appears to be the only or at least the
most important pump which uses CIP as substrate.
[0155] These findings correspond with recent data on triclosan
susceptibility of E. coli which was greatly affected by loss of the
AcrAB pump (McMurry, L. M., et al. 1998. FEMS Microbiol. Lett.
166:305-309). While overexpression of acrAB, marA, or soxS
increased the cells resistance to triclosan about two-fold, a
mutation in the target of triclosan, enoyl reductase (encoded by
fabI), rendered the cell about 100-fold more resistant (McMurry, L.
M., et al. 1998. Nature 394:531-532). However, deletion of acrAB
reduced the resistance 10-fold in all strains, rendering the fabI
mutation less effective, similar to the decrease in effectiveness
of topoisomerase mutations in the case of FQs.
[0156] The prominent finding of this work is that the AcrAB efflux
pump has a powerful role in both the intrinsic and acquired level
of resistance of E. coli to FQs. These data show that efflux
mechanisms decrease the action of Fqs not only in Pseudomonas
(Nikaido, H. 1996. J. Bacteriol. 178:5853-5859) but also in E.
coli, even though in the latter organism the drugs diffuse rapidly
through the more permeable porin channels (Nikaido, H. 1996. J.
Bacteriol. 178:5853-5859). Unidentified mutations in chromosomal
loci in addition to marOR or soxRS modulate the level of resistance
apparently by increasing efflux via AcrAB. Blockage of the AcrAB
efflux pump would increase the potency of drugs such as FQs even in
the face of topoisomerase mutations. TABLE-US-00001 TABLE 1
Fluoroquinolone Resistance Mutations in Escherichia coli AG100 and
its Mar mutant AG112 CIP uptake OFL MIC Fold increase of
Substitution in Mutation marA (% of deenergized (.mu.g/ml)
OFL.sup.a MIC gyrA in marOR expression accumulation) AG100 0.03 --
none none wild-type 51 1-AG100 0.25 8 D87G none wild-type 43
2-AG100 1 32 D87G Frameshift at aa14 overexpression 30 (1485 + 1
bp) 3*-AG100.sup.b 2 64 D87G Asp67Tyr overexpression 9
(1643G.fwdarw.T) 4*-AG100.sup.b 4 128 S83L, D87G Asp67Tyr
overexpression 9 (1643G.fwdarw.T) AG112 0.125 4 -- 5 bp deletion
overexpression 25 (.DELTA.1481-1485) 1-AG112 1 32 D87G 5 bp
deletion overexpression 24 (.DELTA.1481-1485) 2-AG112.sup.c 4 128
D87G 5 bp deletion overexpression 12 (.DELTA.1481-1485)
3-AG112.sup.c 8 256 D87G 5 bp deletion overexpression 9
(.DELTA.1481-1485) .sup.aOfloxacin; Fold change in MIC as compared
to AG100 (wild-type) .sup.bMutants designated with an * were not
derived from 2.degree.- AG100. .sup.cPutative mutation at
additional unknown locus affecting acrAB.
[0157] TABLE-US-00002 TABLE 2 Effects of deletion of acrAB on
susceptibility of fluoroquinolone-resistant mutants of Escherichia
coli AG100 and AG112. MIC.sup.a (.mu.g/ml) OFL.sup.b CIP.sup.b
TVA.sup.b SPX.sup.b Strain acrAB.sup.+ .DELTA.acrAB acrAB.sup.+
.DELTA.acrAB acrAB.sup.+ .DELTA.acrAB acrAB.sup.+ .DELTA.acrAB
AG100 0.03 .ltoreq.0.015 .ltoreq.0.015 .ltoreq.0.015 0.06
.ltoreq.0.03 .ltoreq.0.015 .ltoreq.0.015 1-AG100 0.25 0.06 0.25
0.06 0.25 0.06 0.125 .ltoreq.0.015 2-AG100 1 0.06 0.5 0.06 0.25
0.06 0.25 .ltoreq.0.015 3*-AG100 2 0.06 1 0.06 0.5 0.06 0.5
.ltoreq.0.015 4*-AG100 4 0.125 2 0.125 2 0.125 2 .ltoreq.0.015
AG112 0.125 .ltoreq.0.015 0.06 .ltoreq.0.015 0.125 .ltoreq.0.03
0.06 .ltoreq.0.015 1-AG112 1 0.06 0.5 0.03 0.5 0.06 0.25
.ltoreq.0.015 2-AG112 4 0.06 2 0.03 1 0.06 1 .ltoreq.0.015 3-AG112
8 0.06 4 0.03 2 0.06 2 .ltoreq.0.015 MIC.sup.a (.mu.g/ml) TET.sup.b
CML.sup.b CFOX.sup.6 Strain acrAB.sup.+ .DELTA.acrAB acrAB.sup.+
.DELTA.acrAB acrAB.sup.+ .DELTA.acrAB AG100 1 0.5 4 1 4 0.5 1-AG100
2 1 4 1 4 0.5 2-AG100 2 0.5 16 1 16 0.5 3*-AG100 8 1 32 1 32 0.5
4*-AG100 8 1 64 1 32 1 AG112 4 1 16 1 32 1 1-AG112 4 0.5 32 1 16 1
2-AG112 16 1 64 1 32 0.5 3-AG112 8 0.5 64 1 64 0.5 .sup.aDetermined
as broth microdilution according to NCCLS standards (22).
Representative of experiments done in duplicate. .sup.bOFL,
ofloxacin; CIP, ciprofloxacin; TVA, trovafloxacin; SPX,
sparfloxacin; TET, tetracycline; CML, chloramphenicol; CFOX,
cefoxitin.
[0158] The contents of all references, pending patent applications
and published patents, cited throughout this application are hereby
expressly incorporated by reference.
Equivalents
[0159] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
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