U.S. patent application number 10/205591 was filed with the patent office on 2003-09-11 for methods and compositions for reducing bacterial tolerance to antibacterials, disinfectants and organic solvents.
This patent application is currently assigned to Trustees of Tufts College. Invention is credited to Levy, Stuart B..
Application Number | 20030171317 10/205591 |
Document ID | / |
Family ID | 22032436 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030171317 |
Kind Code |
A1 |
Levy, Stuart B. |
September 11, 2003 |
Methods and compositions for reducing bacterial tolerance to
antibacterials, disinfectants and organic solvents
Abstract
The invention relates to methods and compositions for
manipulating bacterial resistance to non-antibiotic antibacterial
compositions, disinfectants and organic solvents. The invention
provides methods for rendering bacterial cells susceptible to
non-antibiotic antibacterial compositions. Also provided are
methods to reduce the selection of bacterial mutants having an
multiple antibiotic resistance phenotype by non-antibiotic
antibacterial compositions. The invention also provides methods for
testing the ability of non-antibiotic antibacterial compositions to
select for or induce a multiple antibiotic resistance phenotype in
bacteria. Also provided are methods for increasing or decreasing
bacterial tolerance to organic solvents by increasing or decreasing
the activity of bacterial organic solvent efflux pumps.
Compositions useful in the foregoing methods are also provided.
Inventors: |
Levy, Stuart B.; (Boston,
MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Trustees of Tufts College
|
Family ID: |
22032436 |
Appl. No.: |
10/205591 |
Filed: |
July 24, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10205591 |
Jul 24, 2002 |
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09515705 |
Feb 29, 2000 |
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6448006 |
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09515705 |
Feb 29, 2000 |
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08946225 |
Oct 7, 1997 |
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6068972 |
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60060898 |
Oct 3, 1997 |
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Current U.S.
Class: |
514/44A ;
435/252.3; 435/6.13 |
Current CPC
Class: |
C07K 14/245 20130101;
C12Q 1/68 20130101; A61K 38/00 20130101; C12Q 1/18 20130101; C07K
5/06078 20130101; A61P 31/04 20180101 |
Class at
Publication: |
514/44 ;
435/252.3; 435/6 |
International
Class: |
A61K 048/00; C12Q
001/68; C12N 001/21 |
Goverment Interests
[0002] This work was supported in part by U.S. Public Health
Service grant number GM51661. The government may have certain
rights in this invention.
Claims
We claim:
1. A method for inhibiting the selection or propagation of a
bacterial mutant that overexpresses an efflux pump comprising:
contacting bacteria with an agent that binds to a gene locus or an
expression product thereof, wherein the expression of the gene
locus enhances expression of the efflux pump, in an amount
effective to inhibit the gene locus-enhanced expression of the
efflux pump.
2. The method of claim 1, wherein the gene locus is selected from
the group consisting of a mar locus, a sox locus and a rob
locus.
3. The method of claim 2, wherein the gene locus is marA.
4. The method of claim 2, wherein the gene locus is soxS.
5. The method of claim 2, wherein the gene locus is robA.
6. The method of claim 1, wherein the efflux pump is acr-like.
7. The method of claim 6, wherein the efflux pump is acrAB.
8. The method of claim 1, wherein the agent is selected from the
group consisting of antisense nucleic acids, antibodies, ribozymes,
chemicals and proteins which repress expression of the gene
locus.
9. The method of any of claims 1-8, wherein the agent is an
antisense nucleic acids.
10. A method for rendering bacterial cells more susceptible to a
non-antibiotic bactericidal or bacteriostatic agent that is a
substrate of an efflux pump comprising: administering to the
bacterial cell an inhibitor of a gene locus or an expression
product thereof, wherein the expression of the gene locus enhances
expression of an efflux pump.
11. The method of claim 10, wherein the gene locus is selected from
the group consisting of a mar locus, a sox locus and a rob
locus.
12. The method of claim 11, wherein the gene locus is marA.
13. The method of claim 11, wherein the gene locus is soxS.
14. The method of claim 11, wherein the gene locus is robA.
15. The method of claim 10, wherein the efflux pump is
acr-like.
16. The method of claim 15, wherein the efflux pump is acrAB.
17. The method of claim 10, wherein the inhibitor is selected from
the group consisting of antisense nucleic acids, antibodies,
ribozymes, chemicals and proteins which repress expression of the
gene locus.
18. The method of any of claims 10-17, wherein the inhibitor is an
antisense nucleic acid.
19. A method for rendering bacterial cells more susceptible to a
non-antibiotic bactericidal or bacteriostatic agent that is a
substrate of an efflux pump comprising: administering to the
bacterial cell an inhibitor of the efflux pump.
20. The method of claim 19, wherein the efflux pump is
acr-like.
21. The method of claim 20, wherein the efflux pump is acrAB.
22. The method of claim 19, wherein the inhibitor is selected from
the group consisting of
L-phenylalanyl-L-arginyl-.beta.-naphthylamide, 4% ethanol,
methanol, hexane, minocycline.
23. The method of any of claims 19-22, wherein the inhibitor is
L-phenylalanyl-L-arginyl-.beta.-naphthylamide.
24. A method for increasing the ability of bacterial cells to
survive in an organic solvent comprising: enhancing expression in
the bacterial cells of an organic solvent bacterial efflux pump by
growing the bacterial cells in the presence of a non-mar/sox/rob
agent that induces the overexpression of the organic solvent
bacterial efflux pump.
25. The method of claim 24, wherein the agent is a gene encoding an
acr-like pump or an expression product thereof.
26. The method of claim 25, wherein the acr-like pump is acrAB.
27. The method of claim 24, wherein the agent is selected from the
group consisting of an antibiotic, and a non-antibiotic
antibacterial compound.
28. A method for decreasing the ability of bacterial cells to
survive in an organic solvent comprising: reducing expression in
the bacterial cells of an organic solvent bacterial efflux pump by
growing the bacterial cells in the presence of an agent that
reduces the expression of the organic solvent bacterial efflux
pump.
29. The method of claims 28, wherein the agent is an inhibitor of a
gene locus or an expression product thereof, wherein the expression
of the gene locus enhances expression of an efflux pump.
30. The method of claim 29 wherein the gene locus is selected from
the group consisting of a mar locus, a sox locus and a rob
locus.
31. The method of claim 30 wherein the gene locus is marA.
32. The method of claim 30 wherein the gene locus is soxS.
33. The method of claim 30 wherein the gene locus is robA.
34. The method of claim 29 wherein the efflux pump is acr-like.
35. The method of claim 34 wherein the efflux pump is acrAB.
36. The method of claim 29 wherein the inhibitor is selected from
the group consisting of antisense nucleic acids, antibodies,
ribozymes, chemicals and proteins which repress expression of the
gene locus.
37. The method of any of claims 28-36 wherein the inhibitor is an
antisense nucleic acid.
38. A method for testing the ability of a non-antibiotic
composition to induce a multiple antibiotic resistance phenotype in
a bacterium comprising (a) contacting the bacterium with the
non-antibiotic composition, (b) determining the expression of a
bacterial gene locus, the altered expression of which is indicative
of induction of the multiple antibiotic resistance phenotype in the
bacterium, and (c) comparing the result of (b) with a control,
wherein altered expression of the bacterial gene locus indicates
that the non-antibiotic composition induces the multiple antibiotic
resistance phenotype in the bacterium.
39. The method of claim 38, wherein the gene locus is selected from
the group consisting of a mar locus, a sox locus, a rob locus and
an acr-like efflux pump locus.
40. The method of claim 39, wherein the gene locus is marA.
41. The method of claim 39, wherein the gene locus is soxS.
42. The method of claim 39, wherein the gene locus is robA.
43. The method of claim 39, wherein the efflux pump is
acr-like.
44. The method of claim 43, wherein the efflux pump is acrAB.
45. The method of 38, wherein the composition is an inactive
ingredient.
46. The method of claim 45, wherein the inactive ingredient is a
non-bactericidal ingredient.
47. The method of claim 45, wherein the inactive ingredient is a
non-bacteriostatic ingredient.
48. The method of claim 38, wherein step (b) is performed by
determining the enzymatic activity of an expression product of a
marker gene fused to the bacterial gene locus.
49. The method of claim 48, wherein the marker gene is lacZ.
50. A composition comprising: a non-antibiotic bactericidal or
bacteriostatic first agent and a second agent that inhibits the
expression of or activity of an efflux pump.
51. The composition of claim 50, wherein the second agent inhibits
the expression of a gene locus or an expression product thereof,
wherein the expression of the gene locus enhances expression of the
efflux pump.
52. The composition of claim 51, wherein the second agent is
selected from the group consisting of antisense nucleic acids,
antibodies, ribozymes, chemicals and proteins which repress
expression of the gene locus.
53. The composition of claim 52, wherein the second agent is an
antisense nucleic acid.
54. The composition of claim 50, wherein the second agent inhibits
an acr-like efflux pump.
55. The composition of claim 54, wherein the second agent is
selected from the group consisting of
L-phenylalanyl-L-arginyl-.beta.-naphthylamide, 4% ethanol,
methanol, hexane, minocycline.
56. The method of claim 55, wherein the second agent is
L-phenylalanyl-L-arginyl-.beta.-naphthylamide.
57. The composition of claim 50, wherein the first agent is
selected from the group consisting of triclosan, pine oil,
quaternary amine compounds including alkyl dimethyl benzyl ammonium
chloride, chloroxylenol, triclocarbon, disinfectants and organic
solvents.
58. A method for identifying an antibacterial composition which
does not select or induce a multiple antibiotic resistance
phenotype in a bacterium, comprising (a) contacting the bacterium
with the antibacterial composition, (b) determining the expression
of a bacterial gene locus, the altered expression of which is
indicative of induction of the multiple antibiotic resistance
phenotype in the bacterium, and (c) comparing the result of (b)
with a control, wherein a lack of altered expression of the
bacterial gene locus indicates that the antibacterial composition
induces the multiple antibiotic resistance phenotype in the
bacterium.
59. The method of claim 58, wherein the gene locus is selected from
the group consisting of a mar locus, a sox locus, a rob locus and
an acr-like efflux pump locus.
60. The method of claim 59, wherein the gene locus is marA.
61. The method of claim 59, wherein the gene locus is soxS.
62. The method of claim 59, wherein the gene locus is robA.
63. The method of claim 59, wherein the efflux pump is
acr-like.
64. The method of claim 63, wherein the efflux pump is acrAB.
65. The method of claim 58, wherein step (b) is performed by
determining the enzymatic activity of an expression product of a
marker gene fused to the bacterial gene locus.
66. The method of claim 65, wherein the marker gene is lacZ.
Description
RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.119
from PCT application serial number unknown (attorney's docket
number T0359/7007WO), filed Oct. 2, 1997, and U.S. provisional
application serial No. unknown (attorney's docket number
T0359/7007), filed Oct. 3, 1997.
FIELD OF THE INVENTION
[0003] This invention relates to methods and compositions for
manipulating bacterial resistance to non-antibiotic antibacterial
compositions, disinfectants and organic solvents.
BACKGROUND OF THE INVENTION
[0004] Antibiotic or antimicrobial substances have long been used
to inhibit the growth of bacteria or other microbes and to treat
bacterial or microbial infections in humans, other animals, and in
tissue culture. The use of antibiotics or antimicrobials in a
treatment regimen, however, has the undesirable effect of selecting
for bacteria or other microbes which are resistant to those
antibiotics or antimicrobials which are administered or applied. As
a result, treatment regimens can be adversely affected or, in some
cases, rendered ineffective. This necessitates a continual search
for new antibiotics and antimicrobials.
[0005] Of particular interest is the discovery of bacteria which
express a multiple antibiotic resistance phenotype (Mar). This
phenotype entails simultaneous resistance to a multiplicity of
antibiotics which are unrelated in chemical structure. The
appearance of such bacteria and infections by such bacteria greatly
increase the difficulty of identifying effective antibiotics and
treating infections in humans or other animals.
[0006] Multiple antibiotic resistance in bacteria is most commonly
associated with the presence of plasmids and/or transposons which
contain one or more resistance genes, each encoding a single
antibiotic resistance phenotype. Multiple antibiotic resistance
associated with the chromosome, however, has been reported in
Klebsiella, Enterobacter, Serratia (Gutmann et al., J. Infect. Dis.
151:501-507, 1985), Neisseria (Johnson and Morse, Sex. Transm. Dis.
15:217-224, 1988), and Escherichia (George and Levy, J. Bacteriol.
155:531-540, 1983).
[0007] Bacteria expressing a chromosomal multiple antibiotic
resistance phenotype can be isolated by selecting bacteria with a
single antibiotic and then screening for cross-resistance to
structurally unrelated antibiotics. For example, George and Levy
initially described a chromosomal multiple antibiotic resistance
system which exists in Escherichia coli and which can be selected
by a single drug, e.g., tetracycline or chloramphenicol (George and
Levy, 1983). In addition to resistance to the selective agents, the
Mar phenotype includes resistance to structurally unrelated agents,
including nalidixic acid, rifampin, penicillins, and cephalosporins
(George and Levy 1983) as well as fluoroquinolones (Cohen et al.
1989).
[0008] The chromosomal gene locus which correlates with the Mar
phenotype observed in E. coli has been identified. The chromosomal
mar locus, located at 34 min on the E. coli chromosomal map, is
involved in the regulation of intrinsic susceptibility to
structurally unrelated antibiotics (Cohen et al., J. Bacteriol.
175:1484-1492, 1993; Cohen et al., Antimicrob. Agents and
Chemother. 33:1318-1325, 1989; Cohen et al., J. Bacteriol.
170:5416-22, 1988; Goldman et al., Antimicrob. Agents Chemother.
40:1266-1269, 1996), as well as the expression of antioxidant genes
(Ariza et al., J. Bacteriol. 176:143-148, 1994; Greenberg et al.,
J. Bacteriol. 173:4433-4439, 1991) and internal pH homeostasis
(Rosner and Slonczewski, J. Bacteriol. 170:5416-22, 1994). The mar
locus consists of two transcription units (marC and marRAB) which
are divergently transcribed from a central putative
operator-promotor region (marO) (Cohen et al., 1993; Goldman et
al., 1996). marR is the repressor of the marRAB operon (Cohen et
al., 1993; Martin and Rosner, Proc. Natl. Acad. Sci. USA
92:5465-5460, 1995; Seoane and Levy, J. Bacteriol. 177:3414-3419,
1995). Mutations in marR result in increased expression of the
marRAB operon. Overexpression of marA alone is sufficient to
produce the multiple antibiotic resistance phenotype (Cohen et al.,
1993; Gambino et al., J. Bacteriol. 175:2888-2894, 1993; Yan et
al., Abstr. A-26, p. 5, In Abstracts of the 1992 General Meeting of
the American Society for Microbiology, American Society for
Microbiology, Washington, D.C., 1992). marB has no effect of its
own; however, when it is present on the same construct with marA,
it produces a small increase in antibiotic resistance (White et
al., Abst A-104, p. 20. In Abstracts of the 1994 General Meeting of
the American Society for Microbiology, American Society for
Microbiology, Washington, D.C. 1994). The function of marC is
unknown; however, it also appears to enhance the multiple
antibiotic resistance phenotype when cloned on the same DNA
fragment with the marRAB operon (Goldman et al., 1996; White et
al., 1994).
[0009] Overexpression of marA confers multiple antibiotic
resistance via increased efflux of antibiotics, including
fluoroquinolones, tetracycline, and chloramphenicol (Cohen et al.,
1989; George and Levy, 1983; McMurry et al., Antimicrob. Agents
Chemother. 38:542-546, 1994). Transcription of the acrAB operon,
which encodes a multi-drug efflux pump whose expression is
modulated by global stress signals (Ma et al., Mol. Microbiol.
16:45-55, 1995; Ma et al., Mol. Microbiol. 19:101-112, 1996), was
shown to be elevated in strains containing marR mutations and
displaying the Mar phenotype (Okusu et al., J. Bacteriol.
178:306-308, 1996). Moreover inactivation of acrAB led to increased
antibiotic susceptibility in wild type and Mar mutants (Okusu et
al., 1996).
[0010] More recently, mutations of marR have been found in clinical
isolates resistant to quinolones (Maneewannakul and Levy, 1996).
Thus mar mutants can be selected under clinical conditions and not
merely under controlled laboratory conditions. Early mar mutants
(i.e., "first-step" mar mutants) remain susceptible to many common
antibiotics, although such mutants can achieve levels of clinical
resistance to certain antibiotics, including tetracycline,
nalidixic acid and rifampin (reviewed by Alekshun and Levy,
Antimicrob. Agents Chemother. 41:2067-2075, 1997). First-step mar
mutants thus may serve as precursors of bacterial mutants which
display higher levels of resistance resulting from additional
mutations on the chromosome. Thus it has been demonstrated that
antibiotics can select for mutations in chromosomal gene loci which
confer multiple antibiotic resistance under clinical
conditions.
[0011] Non-antibiotic antibacterial compositions such as
disinfectants are widely used in both clinical and consumer
environments for reducing bacterial contamination of work surfaces,
equipment, products and the like. These non-antibiotic
antibacterial compositions have been incorporated into a wide
spectrum of cleansers, disinfectant compositions, soaps, lotions,
plastics, etc. It is not known whether exposure of bacteria to
non-antibiotic antibacterial compositions also can select for
bacterial mutants, including those which display a multiple
antibiotic resistance phenotype.
SUMMARY OF THE INVENTION
[0012] It has now been discovered that bacterial mutants having
multiple antibiotic resistance can be selected by non-antibiotic
antibacterial agents such as common disinfectants. It further has
been discovered that the phenotype of the multiple antibiotic
resistant mutants selected by a non-antibiotic antibacterial agent
results from mutations in chromosomal gene loci which regulate
expression of efflux pumps, which loci have been implicated in
multiple antibiotic resistance phenotypes as described above. The
efflux pumps actively pump out the non-antibiotic antibacterial
agents, as well as organic solvents and antibiotics, thereby
rendering the mutant bacteria resistant to all of the foregoing
compounds.
[0013] According to one aspect of the invention, a method is
provided for inhibiting the selection and/or propagation of a
bacterial mutant that overexpresses an efflux pump. Bacteria are
contacted with an agent that binds to a gene locus (the expression
of the gene locus enhances expression of the efflux pump) or an
expression product thereof, in an amount effective to inhibit the
gene locus-enhanced expression of the efflux pump. In preferred
embodiments, the gene locus is selected from the group consisting
of a mar locus, a sox locus and a rob locus. Also in preferred
embodiments, the efflux pump is acr-like, including the acrAB
efflux pump.
[0014] The agent can be selected from the group consisting of
chemicals, antisense nucleic acids, antibodies, ribozymes, and
proteins which repress expression of the gene locus. A preferred
embodiment is an agent that is an antisense nucleic acid, and in
particularly preferred embodiments, the agent is antisense to the
mar locus, sox locus and/or rob locus. Another preferred embodiment
is chemical inhibitors of efflux pumps, particularly
L-phenylalanyl-L-arginyl-.beta.-naphthylamide.
[0015] According to another aspect of the invention, a method is
provided for rendering bacterial cells more susceptible to a
non-antibiotic bactericidal or bacteriostatic agent that is a
substrate of an efflux pump. An inhibitor of a gene locus or an
expression product thereof is administered to a bacterial cell,
wherein the expression of the gene locus enhances expression of an
efflux pump. In preferred embodiments the gene locus is selected
from the group consisting of a mar locus, a sox locus and a rob
locus. In other preferred embodiments the efflux pump is acr-like
and can be acrAB. The preferred inhibitors are as described
above.
[0016] According to still another aspect of the invention, a method
is provided for rendering bacterial cells more susceptible to a
non-antibiotic bactericidal or bacteriostatic agent that is a
substrate of an efflux pump. The method involves administering to
the bacterial cell an inhibitor of the efflux pump. In preferred
embodiments the efflux pump is acr-like and can be acrAB.
Preferably the inhibitor is selected from the group consisting of
about 4% ethanol, methanol, hexane, minocycline and
L-phenylalanyl-L-arginyl-.beta.-naphthylamide.
[0017] According to another aspect of the invention, a method is
provided for modulating (increasing or decreasing) the ability of
bacterial cells to survive in an organic solvent. In certain
embodiments the method involves enhancing expression in the
bacterial cells of an organic solvent bacterial efflux pump by
growing the bacterial cells in the presence of a non-mar/sox/rob
inducing agent, wherein the agent induces the overexpression of the
organic solvent bacterial efflux pump. The agent can be a gene
encoding an acr-like pump, the acrAB pump, or expression products
thereof. In other embodiments the method involves reducing
expression in the bacterial cells of an organic solvent bacterial
efflux pump by growing the bacterial cells in the presence of an
agent, wherein the agent reduces the expression of the organic
solvent bacterial efflux pump. The agent can be an antisense
nucleic acid which binds to a gene locus encoding an acr-like pump,
especially the acrAB pump, a gene locus which enhances expression
of an efflux pump, such as marA, soxA and robA, and the like. The
agent also can be a ribozyme or a protein which represses
expression of the gene locus. The agent also can be an antibody to
an expression product of the foregoing genes. The agent also can be
a chemical compound which reduces expression of the efflux pump, or
reduces activity of the efflux pump, such as
L-phenylalanyl-L-arginyl-.beta.-naphthylamide.
[0018] According to another aspect of the invention, a method is
provided for testing the ability of a non-antibiotic composition to
induce a multiple antibiotic resistance phenotype in a bacterium.
The bacterium is contacted with the non-antibiotic composition. The
expression of a bacterial gene locus is determined, the altered
expression of which is indicative of induction of the multiple
antibiotic resistance phenotype in the bacterium. Then, the result
of this determination is compared with a control, wherein altered
expression of the bacterial gene locus indicates that the
non-antibiotic composition induces the multiple antibiotic
resistance phenotype in the bacterium. In preferred embodiments,
the gene locus is selected from the group consisting of a mar
locus, a sox locus, a rob locus and an acr-like efflux pump locus.
In one particular embodiment the efflux pump locus is acrAB. The
foregoing methods can be carried out using a non-antibiotic
composition that is an inactive ingredient. The inactive ingredient
can be a non-bactericidal ingredient. The inactive ingredient also
can be a non-bacteriostatic ingredient. In one preferred embodiment
the method is carried out by determining the enzymatic activity of
an expression product of a marker gene, preferably lacZ, fused to
the bacterial gene locus.
[0019] According to another aspect of the invention, a composition
is provided. The composition includes a non-antibiotic bactericidal
or bacteriostatic first agent and a second agent that inhibits the
expression of activity of an efflux pump. In one embodiment, the
second agent inhibits the expression of a gene locus or an
expression product thereof, wherein the expression of the gene
locus enhances expression of the efflux pump. In preferred
embodiments, the second agent is selected from the group consisting
of antisense nucleic acids, antibodies, ribozymes and proteins that
repress expression of the gene locus. In one preferred embodiment
the second agent inhibits an acr-like efflux pump, and particularly
preferred is an antisense nucleic acid. The second agent also can
be selected from the group consisting of 4% ethanol, methanol,
hexane, minocycline and
L-phenylalanyl-L-arginyl-.beta.-naphthylamide. The preferred second
agent is L-phenylalanyl-L-arginyl-.beta.-naphthylami- de. The first
agent in some embodiments is selected from the group consisting of
triclosan, pine oil, quaternary amine compounds including alkyl
dimethyl benzyl ammonium chloride, chloroxylenol, triclocarbon,
disinfectants and organic solvents.
[0020] According to still another aspect of the invention, a method
for identifying an antibacterial composition which does not select
for or induce a multiple antibiotic resistance phenotype in a
bacterium is provided. The bacterium is contacted with the
antibacterial composition. The expression of a bacterial gene locus
is determined, the altered expression of which is indicative of
induction of the multiple antibiotic resistance phenotype in the
bacterium. Then, the result of this determination is compared with
a control, wherein altered expression of the bacterial gene locus
indicates that the antibacterial composition induces the multiple
antibiotic resistance phenotype in the bacterium and a lack of
altered expression of the bacterial gene locus indicates that the
antibacterial composition does not induce the multiple antibiotic
resistance phenotype in the bacterium. In preferred embodiments,
the gene locus is selected from the group consisting of a mar
locus, a sox locus, a rob locus and an acr-like efflux pump locus.
In one particular embodiment the efflux pump locus is acrAB. In one
preferred embodiment the method is carried out by determining the
enzymatic activity of an expression product of a marker gene,
preferably lacZ, fused to the bacterial gene locus.
[0021] The invention also provides methods for identifying
antibacterials which are not subject to efflux pumps, e.g. those
antibacterials which are not substrates for efflux pumps. These
antibacterials are those which have bactericidal or bacteriostatic
action against bacteria which express an efflux pump, particularly
those which overexpress an efflux pump, particularly an acr-like
pump. especially acrAB. These and other aspects of the invention
are described in greater detail below.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 shows the Northern blot analysis of marRAB mRNA in
bacterial mutants.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention is based on the discovery that non-antibiotic
antibacterial compositions and organic solvents select for mutant
bacteria which are resistant not only to the non-antibiotic
antibacterial compositions, but also to a range of antibiotics
(i.e. a multiple antibiotic resistant phenotype) and also to
organic solvents. All of the foregoing compounds are pumped out of
bacteria by efflux pumps, i.e., the foregoing compounds are
substrates for the efflux pumps. Based on these discoveries it is
now possible to enhance the antibacterial properties of
non-antibiotic antibacterial compositions and also reduce the
selection of bacterial mutants having a multiple antibiotic
resistance phenotype by such compositions. The invention also
provides methods for testing the ability of non-antibiotic
antibacterial compositions to select for or induce a multiple
antibiotic resistance phenotype in bacteria. The invention also
provides methods for increasing or decreasing bacterial tolerance
to organic solvents by increasing or decreasing the activity of
bacterial organic solvent efflux pumps, such as by increasing or
decreasing expression of an efflux pump, increasing or decreasing
expression of genes which positively regulate efflux pump gene
loci, and the like. The invention further provides methods for
identifying antibacterial compositions which do not select for or
induce a multiple antibiotic resistance phenotype in bacteria, such
as those antibacterials which are not substrates for efflux pumps.
Compositions useful in the foregoing methods are also provided.
[0024] As used herein, a non-antibiotic antibacterial composition
is a molecule or combination of molecules which are bactericidal or
bacteriostatic, but which are not antibiotics. "Antibiotics" are
those bactericidal or bacteriostatic compounds which are
administered in vivo to people, animals or plants which have a
bacterial infection, or which are used in vitro for research on
bacterial infections of animals. A non-antibiotic antibacterial
composition is not administered to a subject, but rather is used as
a disinfectant for killing bacteria or reducing the growth rate of
a population of bacteria. Non-antibiotic antibacterial compositions
are added as the active ingredients in a variety of industrial and
household disinfectants, such as LYSOL.TM., PINESOL.TM., and the
like. Non-antibiotic antibacterial compositions also are added as
the antibacterial active ingredient in non-disinfectant
compositions such as soaps, lotions, cleansers and the like. More
recently, non-antibiotic antibacterial composition have been
incorporated into plastics for making a variety of articles of
manufacture which have resistance to bacterial growth.
[0025] The non-antibiotic antibacterial compositions, as used
herein, may have active and inactive ingredients. The active
ingredients are, of course, the bactericidal or bacteriostatic
agents which have the effect of slowing or stopping growth of
populations of bacteria, or even killing such populations of
bacteria. Active bactericidal or bacteriostatic ingredients include
triclosan, pine oil, quaternary amine compounds such as alkyl
dimethyl benzyl ammonium chloride, chloroxylenol, triclocarbon, and
other well known disinfectants. The inactive ingredients are the
balance of the components of the non-antibiotic antibacterial
compositions, including surfactants and other cleansing agents,
binders, bulking agents and other compounds. Thus non-antibiotic
antibacterial compositions refers both to the active ingredient of
the compositions as well as the compositions themselves.
[0026] The invention provides methods for inhibiting the selection
or propagation of a bacterial mutant that overexpresses an efflux
pump. By "inhibiting the selection or propagation", it is meant
that the method provides inhibition of selection of a multiple
antibiotic resistant bacterial mutant (i.e., the initial mutation
event which causes the induction of an efflux pump) and/or
inhibition of propagation of a multiple antibiotic resistant
bacterial mutant (i.e., growth and/or replication of such
bacteria).
[0027] The invention also provides methods for rendering bacterial
cells more susceptible to non-antibiotic antibacterial compositions
by administering to the bacterial cells inhibitors of an efflux
pump or a gene locus which enhances expression of the efflux pump,
or an expression product thereof. By "administered to", it is meant
that the bacterial cells are contacted with the inhibitor for a
time sufficient to permit inhibition of the efflux pump or gene
locus.
[0028] The invention further provides methods for increasing or
decreasing organic solvent tolerance of bacterial cells. In these
methods, overexpression of an organic solvent efflux pump is
induced or decreased by growing the cells in the presence of an
agent. By induced "overexpression" it is meant that the organic
solvent efflux pump is expressed at a higher level in bacterial
cells grown in the presence of an inducing agent than in identical
bacterial cells grown under identical conditions but without the
agent, i.e., a level of expression that is sufficient to increase
organic solvent tolerance. By reduced "expression" it is meant that
the organic solvent efflux pump is expressed at a lower level in
bacterial cells grown in the presence of an inhibiting agent than
in identical bacterial cells grown under identical conditions but
without the agent, i.e., a level of expression that is sufficient
to reduce tolerance or increase organic solvent susceptibility.
These methods can also confer organic solvent tolerance or
susceptibility by modulating the activity of an efflux pump as
described herein. Organic solvent tolerance or susceptibility can
be determined by standard methodologies, including those
exemplified in Example 2 below.
[0029] One of the features of antibacterial products is the
reduction in bacterial populations in those products or on those
products, or on surfaces to which such products are applied. As
disclosed herein, non-antibiotic antibacterial products also can
select for multiple antibiotic resistant bacteria. It would be
useful to be able to determine which non-antibiotic antibacterial
compositions select for deleterious mutants. Having determined that
non-antibiotic antibacterial compositions can select for mutants,
it is also possible that other non-antibiotic compositions can
select for mutations. Therefore the invention embraces methods for
testing the ability of non-antibiotic compositions to induce a
multiple antibiotic resistance phenotype. These methods permit
testing of any non-antibiotic composition, including the inactive
ingredients in cleansers, soaps, disinfectants and the like. In
these methods, a bacterial culture is contacted with a
non-antibiotic composition and the expression of a gene locus which
is indicative of a multiple antibiotic resistant phenotype is
determined. The gene locus expression can be determined by any
convenient method, of which many are known in the art. These
methods include enzyme assays comprising fusions of regulatory loci
to a marker gene (e.g. as described for a mar regulatory locus in
PCT published application WO94/05810), amplification of gene
transcripts (such as using polymerase chain reaction),
hybridization methods including Northern blots, and measurement of
protein expression including Western blots, ELISA, etc. The level
of expression of the gene locus is then compared with a control to
determine if the non-antibiotic compositions induced the multiple
antibiotic resistant phenotype.
[0030] According to the invention, various agents which inhibit the
expression or activity of an efflux pump or gene loci which control
expression of the efflux pump are useful for reducing selection
and/or propagation of mutant bacteria, and also render the cells
more susceptible to non-antibiotic antibacterial compositions.
These inhibitors are contacted with or administered to the
bacterial cells to prevent the undesirable effects of the
non-antibiotic antibacterial compositions. One convenient way to
ensure contact of the appropriate bacterial cell populations is to
include the inhibitors and agents in the non-antibiotic
antibacterial compositions. Thus the invention further provides
compositions comprising a non-antibiotic bactericidal or
bacteriostatic first agent and a second agent which inhibits the
expression or activity of an efflux pump, as described above. These
compositions can be prepared according to the standard procedures
used to prepare non-antibiotic antibacterial compositions. For
example, a standard disinfectant composition such as PINE-SOL.TM.
can have added to it an effective amount of an inhibitor of an
efflux pump such as described in PCT published patent application
WO96/33285, or an antisense nucleic acid which binds to the efflux
pump gene locus, etc.
[0031] By "effective amount" is meant an amount of the second agent
which reduces the selection of mutants by the non-antibiotic first
agent. Effective amounts can be determined using standard bacterial
growth and mutation assays, including those provided herein. For
example, various amounts of the second agent can be added to a
non-antibiotic antibacterial composition, and the combined
composition can be used as provided in the examples below to select
bacterial mutants. Any amount of the second agent which reduces the
number of mutants selected relative to the number of mutants
selected by the non-antibiotic antibacterial composition alone is
an effective amount. One of ordinary skill in the art can determine
with no more than routine experimentation what constitutes an
effective amount of a second agent, and what amount of a second
agent is optimal to prevent selection of mutants by the
non-antibiotic antibacterial compositions. Effective amounts of
other inhibitors and agents disclosed herein can be determined
similarly.
[0032] As disclosed herein, inhibitors of the marA gene locus and
other loci which regulate efflux pumps are effective to reduce the
selection of antibiotic resistant bacterial mutants by
non-antibiotic antibacterial compositions, and also potentiate the
antibacterial properties of such compositions. The marA gene has
been cloned and sequenced, the sequence deposited as GenBank
accession number M96235. The marA gene has homologs in E. coli, as
well as in other species of bacteria. Inhibitors of such marA
homologs also are useful for reducing the selection of antibiotic
resistant bacterial mutants and potentiating the antibacterial
properties of non-antibiotic antibacterial compositions.
[0033] For example, the MarA protein is homologous to both SoxS,
the effector of the soxRS regulon (Fawcett and Wolf, Mol.
Microbiol. 14:669-679, 1994; Li and Demple, J. Biol. Chem.
269:18371-18377, 1994), and RobA, a small protein that binds to the
E. coli replication origin and some stress gene promoters (Ariza et
al., 1995; Cohen et al., 1995; Jair et al., J. Bacteriol.
178:2507-2513, 1996; Skarstad et al., J. Biol. Chem. 268:5365-5370,
1993). The soxRS regulon mediates the cell's response to oxidative
stress (Amabile-Cuevas and Demple, Nucleic Acids Res. 19:4479-4484,
1991; Nunoshiba et al., J. Bacteriol. 174:6054-6060, 1992; Wu and
Weiss, J. Bacteriol. 173:2864-2871, 1991). soxS genes include those
found in S. typhimurium (GenBank accession number U61147) and E.
coli (GenBank accession numbers X59593 and M60111). robA genes
include those found in E. coli (GenBank accession numbers AE000509,
U00096, M97495 and M94042).
[0034] Other known homologs of marA include those found in
Enterobacteriaceac by nucleic acid hybridization under stringent
conditions (Cohen et al., 1993). Other marA homologs include pqrA,
identified in Proteus vulgaris (GenBank accession number D13561),
ramA identified in Klebsiella pneumonia (GenBank accession number
U19581), and aarP identified in Providencia stuartii (GenBank
accession number L38718).
[0035] Additional homologs of marA (and other gene loci useful
according to the invention) can be identified by conventional
techniques. Such techniques include cloning by hybridization to
marA or to known homologs thereof, and functional cloning. Cloning
by hybridization involves subjecting marA or known homologs thereof
to hybridization with nucleic acids of bacteria (preferably the
chromosomal DNA) under stringent conditions. The term "stringent
conditions" as used herein refers to parameters with which the art
is familiar. Nucleic acid hybridization parameters may be found in
references which compile such methods, e.g. Molecular Cloning: A
Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or
Current Protocols in Molecular Biology, F. M. Ausubel, et al.,
eds., John Wiley & Sons, Inc., New York. More specifically,
stringent conditions, as used herein, refers, for example, to
hybridization at 65.degree. C. in hybridization buffer
(3.5.times.SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02%
Bovine Serum Albumin, 2.5 mM NaH.sub.2PO.sub.4(pH7), 0.5% SDS, 2 mM
EDTA). SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDS
is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic
acid. After hybridization, the membrane upon which the DNA is
transferred is washed at 2.times.SSC at room temperature and then
at 0.1.times.SSC/0.1.times.SDS at temperatures up to 65.degree.
C.
[0036] There are other conditions, reagents, and so forth which can
be used, which result in a similar degree of stringency. The
skilled artisan will be familiar with such conditions, and thus
they are not given here. It will be understood, however, that the
skilled artisan will be able to manipulate the conditions in a
manner to permit the clear identification of homologs and alleles
of nucleic acids of the invention. The skilled artisan also is
familiar with the methodology for screening cells and libraries for
expression of such molecules which then are routinely isolated,
followed by isolation of the pertinent nucleic acid molecule and
sequencing.
[0037] In general homologs typically will share at least 30%
nucleotide identity and/or at least 40% amino acid identity to
mar/sox/rob genes or to efflux pumps genes, or their polypeptide
products respectively, in some instances will share at least 50%
nucleotide identity and/or at least 65% amino acid identity and in
still other instances will share at least 60% nucleotide identity
and/or at least 75% amino acid identity. Watson-Crick complements
of the foregoing nucleic acids also are embraced by the
invention.
[0038] Functional cloning is useful to isolate homologs which do
not share sufficient homology at the nucleotide or amino acid
sequence level to permit cloning by nucleic acid hybridization, but
which nevertheless are functional equivalents of the genes useful
in the invention. Functional equivalents need not exhibit the same
level of activity, merely activity of the same kind. For example,
one phenotypic manifestation of mara expression is the induction of
the expression of a set of genes, including acrA. A gene which
induces substantially the same set of genes but at a lower level of
expression would be considered a functional equivalent.
[0039] Functional cloning, as used herein, involves expression of a
nucleic acid sequence in a bacterium and determining whether the
expression of that sequence confers a desired phenotype on the
bacterium. It is known that marA homologs exhibit similar
functional characteristics with respect to multiple antibiotic
resistance phenotype. For example, overexpression of either soxS or
robA in E. coli produces both increased organic solvent tolerance
and low-level resistance to multiple antimicrobial agents (Ariza et
al., J. Bacteriol. 177:1665-1661, 1995; Nakajima et al., Biosci.
Biotechnol. Biochem. 59:1323-1325, 1995a; Nakajima et al., Appl.
Environ. Microbiol. 61:2302-2307, 1995b). Thus, for marA homologs,
the desired phenotype can be multiple antibiotic resistance,
induction of mar-regulated genes (see, e.g., U.S. Pat. No.
5,650,321), and the like. For determining multiple antibiotic
resistance, all that is necessary is to express the putative marA
homolog in a non-multiple antibiotic resistant bacterium and
determine whether the modified bacterium acquires resistance to
more than one antibiotic, such as tetracycline, chloramphenicol,
nalidixic acid, etc. marA homologs can be expressed according to
standard procedures, such as transformation with an expression
plasmid containing the marA homolog, introduction of one or more
copies of the marA homolog on the bacterial chromosome via
transposon-mediated insertion, etc.
[0040] The acrAB locus, positively regulated by MarA (Ma et al.,
Mol. Microbiol. 16:45-55, 1995) and SoxS and RobA (Ma et al., Mol.
Microbiol. 19:101-112, 1996), specifies a
proton-motive-force-dependent multidrug efflux pump for a wide
variety of mostly lipophilic substances (Ma et al., 1995; Nikaido,
Bacteriol. 178:5853-5859, 1996; Nikaido, Science 264:382-387, 1994;
Paulsen et al., Microbiol Rev. 60:575-608, 1996). Mar mutants and
wild type strains deleted of this locus become equally
hypersusceptible to antibiotics (Okusu et al., J. Bacteriol.
178:306-308, 1996) suggesting that the acrAB pump confers an
intrinsic resistance level which is then enhanced in Mar
mutants.
[0041] The acrA and acrB genes have been cloned and sequenced. For
example, the sequences of acrAB in E. coli are deposited as GenBank
accession number U00734. The acrAB genes have homologs in E. coli,
as well as in other species of bacteria. Sequence homologs of acrAB
efflux pumps are referred to herein as "acr-like" efflux pumps.
Isolation of acr-like efflux pumps and other efflux pumps can be
carried out according to the methods described above for nucleic
acid hybridization and functional cloning. Inhibitors of such acrAB
homologs also are useful for reducing the selection of antibiotic
resistant bacterial mutants and potentiating the antibacterial
properties of non-antibiotic antibacterial compositions.
[0042] Agents which induce overexpression of acr-like efflux pumps
are useful in promoting organic solvent tolerance. Inducers of
efflux pumps include genes which encode the various efflux pumps
which when expressed in a bacterium as a nucleic acid operably
linked to a promoter can increase the numbers of efflux pump
protein molecules in the bacterium. Agents also include molecules
which inhibit the function of efflux pump regulatory genes. For
example, antisense nucleic acids which bind to acrR and prevent its
transcription or translation would function as inducers of acrAB.
Efflux pumps can also be induced by mutation of regulatory genes
(such as acrR for the acrAB pump).
[0043] Agents useful in decreasing the expression or activity of an
efflux pump for increasing organic solvent susceptibility
(decreasing organic solvent tolerance) are provided in the
following paragraphs.
[0044] Agents which bind to a gene locus which mediates enhanced
expression of an efflux pump (such as the mar/sox/rob class of
genes) or a nucleic acid expression product thereof include
antisense nucleic acids, ribozymes and regulatory proteins such as
repressor proteins (e.g. MarR). For example, antisense nucleic
acids which bind to marA and prevent transcription or translation
thereof would function as inhibitors of marA and agents which bind
marA. Agents which bind to a protein expression product of a gene
locus include antibodies. Inhibitors of the foregoing gene loci and
expression products also include molecules which bind to the gene
loci and expression products as described above. Other classes of
agents and inhibitors of these types will be known to those of
skill in the art.
[0045] Classes of inhibitors of efflux pumps useful in the methods
and compositions of the invention have been described previously in
PCT published patent application WO96/33285 (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. For example, antisense nucleic
acids which bind to acrAB genes and prevent transcription or
translation thereof would function as inhibitors of acrAB.
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 reduces the expression of efflux
pumps in bacteria.
[0046] 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).
As used herein, the term "antisense oligonucleotide" or "antisense"
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 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.
[0047] In one set of embodiments, the antisense oligonucleotides of
the invention may be composed of "natural" 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.
[0048] 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 which do not prevent them from hybridizing to
their target but which enhance their stability or targeting or
which otherwise enhance their therapeutic effectiveness.
[0049] 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.
[0050] 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,
contemplates preparations containing modified antisense molecules
that are complementary to and hybridizable with, under
physiological conditions, nucleic acids encoding mar/sox/rob or
efflux pump polypeptides, together with one or more carriers.
[0051] 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. Antibodies include
polyclonal and monoclonal antibodies, prepared according to
conventional methodology.
[0052] Significantly, as is well-known in the art, only a small
portion of an antibody molecule, the paratope, is involved in the
binding of the antibody to its epitope (see, in general, Clark, W.
R. (1986) The Experimental Foundations of Modern Immunology Wiley
& Sons, Inc., New York; Roitt, I. (1991) Essential Immunology,
7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and
Fc regions, for example, are effectors of the complement cascade
but are not involved in antigen binding. An antibody from which the
pFc' region has been enzymatically cleaved, or which has been
produced without the pFc' region, designated an F(ab').sub.2
fragment, retains both of the antigen binding sites of an intact
antibody. Similarly, an antibody from which the Fc region has been
enzymatically cleaved, or which has been produced without the Fc
region, designated an Fab fragment, retains one of the antigen
binding sites of an intact antibody molecule. Proceeding further,
Fab fragments consist of a covalently bound antibody light chain
and a portion of the antibody heavy chain denoted Fd. The Fd
fragments are the major determinant of antibody specificity (a
single Fd fragment may be associated with up to ten different light
chains without altering antibody specificity) and Fd fragments
retain epitope-binding ability in isolation. Any of the foregoing
antigen fragments are useful in the methods and compositions of the
invention. The present invention also includes so-called single
chain antibodies and intracellular antibodies.
EXAMPLES
Example 1
[0053] Mutants resistant to Pine-Sol/pine oil were obtained from
stationary phase LB broth cultures of E. coli strain "WEC" (wild
type strain 15-5068 from Carolina Biological Supply Co.,
Burlington, N.C.) and AG100 (George and Levy, J. Bacteriol.
155:531-540, 1983), at 30.degree. C. on nutrient agar (NP 3.5 GP)
or LB agar with 2-3 days incubation in a variety of ways: using a 6
mm disc method; plating cells on plates or gradient plates (Curiale
and Levy, J. Bacteriol. 151:209-215, 1982), containing PINE-SOL.TM.
(product of Clorox Co., Oakland, Calif.) or pine oil itself.
[0054] Antibiotic susceptibility was measured at 30.degree. C.
using antibiotic susceptibility discs (Carolina Biological),
gradient plates with the drug in the top agar (Curiale and Levy,
1982) or agar dilution plates (concentration steps of 1.5 fold;
inocula of 10.sup.5 cells/5 .mu.l spot). While there was a
variability of resistance phenotypes, all Pine-Sol/pine
oil-selected mutants were also multidrug resistant (Table 1 A,
B).
1 TABLE 1A Susceptibility by discs; diameter of clearing (mm)
Strain Characteristics Ap Cm Tc WEC wild type 22 27 21 NP3.5GP
mutant of WEC selected on Pine- 12 11 14 Sol gradient (0-1.5%)
[0055]
2 TABLE 1B Susceptibility by gradient plates (MIC).sup.b (% by
volume) (.mu.g/ml) Strain Characteristics PS Ap Cm Nal Tc AG100
wild type 0.9 <1.2 2.6 1.7 1.8 AP1 mutant of AG100 >3.6/0.9
3.0 7.8 9.7 2.4 selected by pine oil on disc AP5 mutant of AG100
>2.9/0.9 7.2 21 7.5 4.5 selected as for AP1 APS3 mutant of AG100
1.8 7.7 >35 8.6 5.3 selected on Pine- Sol gradient (0-1.5%)
AG102 [ 1 ].sup.d Mar mutant of >4.1 8.5 >35 14.0 >12.8
AG100, selected on Tc (2 steps) HH180 [ 2 ].sup.d deletion of 39 kb
0.3 <0.6 ND.sup.c <1.8 <0.6 including mar locus; has zdd-
230::Tn9 (Cm.sup.R); in host strain MM294 HH188 [ 2 ].sup.d HH180
containing 0.9 <1.0 ND.sup.c 3.7 1.2 pHHM183 (mar+) HH191 [ 2
].sup.d HH180 containing 2.3 5.4 ND.sup.c 9.1 8.2 pHHM191 (marR2)
HH193 [ 2 ].sup.d HH180 containing 3.2 5.9 ND.sup.c 10.9 >11.4
pHHM193 (marR5) [ 3 ].sup.d .sup.aAbbreviations: PS (Pine-Sol), Ap
(ampicillin), Cm (chloramphenicol), Nal (nalidixic acid), Tc
(tetracycline). .sup.bGradient plate values were the averages of
two to four experiments, except in the case of chloramphenicol,
which involved a single determination. .sup.cHost strain is CmR due
to Tn9, so values were not determined (ND). .sup.dReferences: 1)
George et al., 1983; 2) Cohen et al., 1993; 3) Seoane and Levy,
1995
[0056] In host strain HH180, deleted of the entire mar region,
plasmid pHHM188, bearing a 9 kb wild type mar caused no change in
the resistance phenotype. In the same host pHHM191 and pHHM193 each
containing cloned 9 kb fragment including the entire mar locus, and
marR was mutant, caused a Mar phenotype (Cohen et al., J.
Bacteriol. 175:1484-1492, 1993). These Mar mutants, as well as
AG102 were resistant to Pine-Sol (Table 1B) and to 100% pine oil
when compared to their respective wild type strains.
3TABLE 2 Effect of inactivation of mar, sox, rob, or acr locus upon
susceptibility to Pine-Sol Relative MIC for Pine-Sol.sup.a Strain
mar.sup.b sox.sup.b rob.sup.b acr.sup.b AG100 1 0.9 0.8 <0.06
AP1 0.5 <0.6 0.5 <0.02 AP5 0.4 0.9 0.8 <0.02 APS3 0.4 1 1
<0.04 AG102 0.4 1 1 <0.03 .sup.aRelative MIC is the MIC of
the inactivated strain divided by the MIC of the strain before
inactivation. Values in bold face indicate notable increases in
susceptibility. Values obtained from both gradient plate and agar
dilution experiments were averaged. .sup.bInactivated locus
[0057] Northern blot analysis for expression of marA mRNA using a
[radiolabeled] marA probe in the absence and presence of the
inducer salicylate (Cohen et al., J. Bacteriol. 175:7856-7862,
1993) revealed that, like Mar mutant AG102, mutants AP5 and NP3.5
GP showed an over expression of marA that was enhanced by
salicylate (FIG. 1). Over expression was also seen in mutant APS3
(data not shown). The wild type AG100 and the pine oil mutant AP1
showed no detectable signal (FIG. 1). We concluded that AP5,
NP3.5GP, and APS3, but not AP1, were probably Mar mutants.
[0058] The marCORAB locus was deleted in the Pine-Sol/pine oil
mutants and in AG102 by P1 transduction (Provence and Curtiss, p.
317-347, In Gerhardt et al., eds., Methods for General and
Molecular Bacteriology. ASM, Washington, D.C., 1994) using
AG100/Kan (Maneewannakul and Levy, 1996) as the donor strain and
selecting on kanamycin. The deletion caused a 60-70% reduction in
the resistance of mutants to Pine-Sol (Table 2), down to
approximately a wild type level. The same was true for mutant
NP3.5GP (data not shown).
[0059] Inactivation of the soxRS and robA loci in the Pine-Sol/pine
oil and Mar mutants via P1 transduction of a kanR gene in the gene
caused decreased resistance to Pine-Sol only in the mutant AP1
(Table 2). Mutant AP1 did not overexpress mar or soxRS (data not
shown). Deletion of the acrAB locus by P1 transduction of kanR in
the gene increased susceptibility to Pine-Sol in all strains (Table
2).
[0060] Deletion of acrAB (but not of mar) also caused more than a
ten fold increase in the susceptibility of strains to the products
containing the quaternary amine or chloroxylenol (data not shown),
suggesting that AcrAB was also involved in effluxing those two
disinfectants.
Example 2
[0061] Organic solvent tolerance mediated by the marA, soxS, robA
and acrAB loci of E. coli is described in White et al. (J.
Bacteriol. 179:6122-6126, 1997). These results are summarized
below. AG102, a Mar mutant of AG100, grew in the presence of
n-hexane, cyclohexane (Table 3), and n-pentane (data not shown)
whereas AG100 grew only in hexane.
4TABLE 3 Organic solvent tolerancc of wild-type and mar strains
bearing mar, soxS, or robA plasmids. Growth in presence of organic
solvent.sup.a Strain Plasmid.sup.b n-hexane (3.9).sup.c cyclohexane
(3.4) AG102 (marR ++ ++ mutation) AG100 (wild-type) ++ - AG100
pMAK-TU1 ++ - AG100 pMAK-TU2 ++ + AG100 pMAK-TU1 & ++ ++ TU2
AG100 pSMarAB ++ + AG100 pSXS ++ ++ AG100 pSRob ++ ++ AG100K + -
(marCORAB::kan) AG100K pMAK-TU1 + - AG100K pMAK-TU2 ++ + AG100K
pMAK-TU1 & ++ ++ TU2 AG100K pSMarAB ++ + AG100K pSXS ++ ++
AG100K pSRob ++ ++ MCH164 (.DELTA.mar) + - MCH164 pMAK-TU1 + -
MCH164 pMAK-TU2 ++ - MCH164 pMAK-TU1 & ++ ++ TU2 MCH164 pSMarAB
++ - MCH164 pSXS ++ - MCH164 pSRob ++ - AG100-B (acrR ++ + mutant)
AG100-A (.DELTA.acrAB) - - AG102-A (marR1, - - .DELTA.acrAB)
AG102-A pSMarAB - - AG102-A pSXS - - AG102-A pSRob - - .sup.a(++)
signifies confluent growth; (+) visible growth(.ltoreq.100
colonies); (-) signifies no growth. .sup.bIPTG was added to plates
at a concentration of 0.5 mM when induction of plasmid genes was
required (pSE380 derivatives). .sup.cValues in parentheses are log
Pow.
[0062] In the wild type E. coli AG100 background, over expression
of marA (on plasmid pSMarAB or pMAK-TU2) or soxS (on pSXS) or robA
(on pSRob) resulted in cyclohexane tolerance (Table 3). marC by
itself (pMAK-TU1) had no effect on cyclohexane tolerance, however,
introduction of marCORAB on the low copy plasmid pMAK705 (pMAK-TU1
& TU2) resulted in cyclohexane tolerance (Table 3).
[0063] When the mar locus was inactivated by replacement with a
kanamycin resistance cassette (AG100K) (Maneewannakul and Levy,
1996), the strain became hypersusceptible to n-hexane as compared
to the wild type strain (Table 3). MCH164 [a derivative of AG100
from which 39 kb of chromosomal DNA including the entire mar locus
had been deleted (Goldman et al., Antimicrob. Agents Chemother.
40:1266-1269, 1996; McMurry et al., Antimicrob. Agents Chemother.
38:542-546, 1994)] was, as expected, also hypersusceptible to
organic solvents (Table 3). Expression in trans of marA, soxS, or
robA in AG100K, restored n-hexane tolerance, and increased
cyclohexane tolerance in the cell (Table 3). Expression in trans in
AG100K of marA, specified from plasmid pMAK-TU1 & TU2 restored
n-hexane tolerance and produced higher cyclohexane tolerance (Table
3). While introduction of either marA, soxS, or robA restored
n-hexane tolerance in MCH164, only pMAK-TU1 TU2 produced
cyclohexane tolerance in this larger deletion mutant (Table 3).
[0064] Overexpression of acrAB, because of a mutation in acrR in
AG100-B, enabled the strain to grow in the presence of cyclohexane
(Table 3). Deletion of acrAB from wild-type AG100 (AG100-A)
resulted in n-hexane sensitivity (Table 3). Deletion of acrAB from
the Mar mutant (AG102-A) resulted in both n-hexane and cyclohexane
sensitivity. Expression of marA, soxS, or robA in AG102-A failed to
restore organic solvent tolerance, further demonstrating the
critical role of acrAB (Table 3).
[0065] E. coli strains overexpressing MarA (JHC1069; cfxB1/MarR
mutation) or SoxS (JTG1078; soxR105 mutation) grew in the presence
of both n-hexane and cyclohexane, whereas the wild-type C4468 only
grew in the presence of n-hexane (Table 4). Much like the situation
in AG100, introduction of either pSMarAB, pMAK-TU2, pMAK-TU1 &
TU2, pSXS, or pSRob into GC4468 produced cyclohexane tolerance.
Inactivation of robA by insertion of a kanamycin cassette (RA4468)
caused n-hexane susceptibility (Table 4). Introduction of either
marA (on pMAK-TU1 & TU2, pMAK-TU2, or pSMarAB), SoxS (on pSXS),
or RobA (on pSRob), into the robA inactivated strain, increased
both n-hexane and cyclohexane tolerance (Table 4). Deletion of
soxRS (DJ901) had little effect on n-hexane tolerance (Table 4).
Introduction of marA, soxS, or robA into the .DELTA.soxRS strain
produced cyclohexane tolerance (Table 4). In all these
complementations, the effect of marA was best noted from plasmid
pMAK-TU1 & TU2.
5TABLE 4 Organic solvent tolerance of wild-type, .DELTA.soxRS, or
robA::Kan strains bearing mar, soxS, or robA plasmids. Growth in
presence of organic solvent.sup.a Strain Plasmid.sup.b n-hexane
(3.9).sup.c cyclohexane (3.4) GC4468 (wild-type) ++ - JHC1069
(cfxB1) ++ ++ JTG1078 (soxR105) ++ ++ GC4468 pMAK-TU1 ++ - GC4468
pMAK-TU2 ++ + GC4468 pMAK-TU1 & ++ ++ TU2 GC4468 pSMarAB ++ +
GC4468 pSXS ++ ++ GC4468 pSRob ++ ++ RA4468 (robA::kan) + - RA4468
pMAK-TU1 + - RA4468 pMAK-TU2 ++ + RA4468 pMAK-TU1 & ++ ++ TU2
RA4468 pSMarAB ++ + RA4468 pSXS ++ ++ RA4468 pSRob ++ ++ DJ901
(AsoxRS) ++ - DJ901 pMAK-TU1 ++ - DJ901 pMAK-TU2 ++ + DJ901
pMAK-TU1 & ++ ++ TU2 DJ901 pSMarAB ++ + DJ901 pSXS ++ ++ DJ901
pSRob ++ ++ .sup.a(++) signifies confluent growth; (+) visible
growth(.ltoreq.100 colonies); (-) signifies no growth. .sup.bIPTG
was added to plates at a concentration of 0.5 mM when induction of
plasmid genes was required (pSE380 derivatives). .sup.cValues in
parenthese are log Pow.
[0066] These results show that overexpression of marA, soxS, or
robA leads to increased organic solvent tolerance and that
tolerance is mediated by the acrAB efflux pump.
[0067] Equivalents
[0068] 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.
[0069] All references disclosed herein are incorporated by
reference.
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