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Antimicrobial Agents and Chemotherapy, October 2001, p. 2703-2709, Vol. 45, No. 10
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.10.2703-2709.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Enhancement of Fluoroquinolone Activity by
C-8 Halogen and Methoxy Moieties: Action against a Gyrase Resistance
Mutant of Mycobacterium smegmatis and a Gyrase-Topoisomerase
IV Double Mutant of Staphylococcus aureus
Tao
Lu,1
Xilin
Zhao,1
Xinying
Li,1
Alex
Drlica-Wagner,1
Jian-Ying
Wang,1
John
Domagala,2 and
Karl
Drlica1,*
Public Health Research Institute, New York,
New York 10016,1 and Parke-Davis
Research Division, Warner-Lambert Company, Ann Arbor, Michigan
481052
Received 4 December 2000/Returned for modification 10 January
2001/Accepted 6 July 2001
 |
ABSTRACT |
The increasing prevalence of antibiotic resistance among bacterial
pathogens prompted a microbiological study of fluoroquinolone structure-activity relationships with resistant mutants. Bacteriostatic and bactericidal activities for 12 fluoroquinolones were examined with
a gyrase mutant of Mycobacterium smegmatis and a
gyrase-topoisomerase IV double mutant of Staphylococcus
aureus. For both organisms C-8 halogen and C-8 methoxy
groups enhanced activity. The MIC at which 99% of the isolates tested
were inhibited (MIC99) was reduced three- to fivefold for
the M. smegmatis mutant and seven- to eightfold for the
S. aureus mutant by C-8 bromine, chlorine, and methoxy
groups. With both organisms a smaller reduction in the
MIC99 (two- to threefold) was associated with a C-8
fluorine moiety. In most comparisons with M. smegmatis the
response to a C-8 substituent was similar (within twofold) for
wild-type and mutant cells. In contrast, mutant S. aureus
was affected more than the wild type by the addition of a C-8
substituent. C-8 halogen and methoxy groups also improved the ability
to kill the two mutants and the respective wild-type cells when
measured with various fluoroquinolone concentrations during an
incubation period equivalent to four to five doubling times.
Collectively these data help define a group of fluoroquinolones that
can serve (i) as a base for structure refinement and (ii) as test
compounds for slowing the development of fluoroquinolone resistance
during infection of vertebrate hosts.
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INTRODUCTION |
Antimicrobial resistance is now well
documented for many pathogens, and studies with a variety of bacteria
indicate that resistance can develop within just a few years (1,
3, 13, 19, 25). As an antimicrobial agent becomes ineffective,
it tends to be replaced by another agent; then, resistance develops to
the second agent. For example, in the mid-1990s many isolates of
Streptococcus pneumoniae exhibited resistance to penicillin
(1). The fluoroquinolones ciprofloxacin and levofloxacin
were substituted for penicillin, and now fluoroquinolone resistance is
appearing (6). Preserving existing agents may require
interruption of the process by which resistance develops to one agent
after another.
One approach for slowing the development of resistance is to identify
and use derivatives that exhibit preferential activity against
resistant mutants (31). Such a feature has been seen with
fluoroquinolones, initially with derivatives containing C-8 chlorine
and C-8 methoxy moieties (17, 20). These observations suggested a reason that sparfloxacin, the first C-8-substituted fluoroquinolone in clinical use, is more active against resistant Mycobacterium tuberculosis than the C-8 hydrogen compound
ciprofloxacin (8, 27). Subsequent comparisons between C-8
methoxy and C-8 hydrogen derivatives strengthened the observation that
activity against resistant mutants can be improved preferentially
(8, 9, 31).
In the present work we examined a set of fluoroquinolones for activity
against two resistant mutants to rank the C-8 moieties for activity
enhancement. Mycobacterium smegmatis was chosen as a
representative of organisms thought to have only gyrase as the fluoroquinolone target (7), while Staphylococcus
aureus represented those having both gyrase and topoisomerase IV
as targets (11, 12). In most cases the C-8-substituted
fluoroquinolones blocked mutant growth and killed mutants better than
comparable C-8 hydrogen compounds. Enhancement of activity was usually
greater with a C-8 methoxy group and lower with a C-8 fluorine moiety.
Structural similarities, plus enhanced activity against resistant
mutants, characterize C-8 halogen and methoxy compounds as members of a distinct group of fluoroquinolones.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
M.
smegmatis mc2155 and its gyrA
quinolone-resistant mutant KD2003 have been described previously
(32). Strain KD2003, which contains an Asp-95-to-Gly
substitution in GyrA, was selected as a first-step mutant on agar
containing 13 times the MIC at which 99% of isolates tested are
inhibited (MIC99) of PD160788, a C-8 hydrogen
fluoroquinolone (32). M. smegmatis was grown as
described previously (18). Incubation was at 37°C. The
doubling time in 7H9 medium was 3.5 to 4 h for both strains.
S. aureus strain ISP794 (4) and its gyrA
parC (grlA)-resistant mutant EN1252 were obtained from
David Hooper (Massachusetts General Hospital, Boston). The construction
of EN1252, which contains a Ser-84-to-Leu substitution in GyrA and a
Ser-80-to-Phe substitution in ParC (Gr1A), was described in reference
(4). S. aureus strains were cultured with
vigorous shaking at 37°C in CY medium (24). The doubling
time in CY medium was 25 min.
Fluoroquinolones.
The fluoroquinolones used (and their
sources) were as follows: ciprofloxacin (Miles Laboratories, Kankakee,
Ill.; currently available from Bayer AG, West Haven, Conn.);
moxifloxacin and Bay y 3114 (Bayer AG) and sparfloxacin and
investigational compounds (Parke-Davis Pharmaceutical Co., Ann Arbor,
Mich.). Fluoroquinolone solutions were prepared as described in
reference (26). Agar plates containing fluoroquinolone
were prepared by adding concentrated solutions to molten agar.
Measurement of bacteriostatic and bactericidal activity.
To
measure MIC99s, stationary phase cells, prepared by
overnight growth, were diluted using liquid medium, and 10-µl
aliquots of each dilution were spotted on quinolone-containing agar
plates. Colonies on each plate were counted after incubation at 37°C
for 1 day (S. aureus) or 2 to 3 days (M. smegmatis). MIC99 was defined as the
fluoroquinolone concentration required to inhibit colony formation by
99%. Preliminary determinations using twofold dilutions of
fluoroquinolone provided an approximate value of MIC99.
This measurement was followed by a second measurement, plus a
replicate, that utilized linear drug concentration increments that
increased by 10 to 20% at each step. Numbers of colonies recovered
were plotted against drug concentration to determine the
MIC99 by interpolation.
To measure bactericidal activity, small aliquots of frozen cultures
were introduced into liquid medium to give a cell density
of
10
7 CFU/ml. Cells were then grown to exponential phase
(10
8 CFU/ml) by shaking at 37°C. Cultures were
distributed into tubes
containing liquid medium and various
concentrations of fluoroquinolone
(see Fig.
2 through
5). Incubation
with shaking was continued
for either 2 h (
S. aureus)
or 18 h (
M. smegmatis). Serial dilutions,
which
eliminated drug carryover, were prepared, and aliquots from
the
dilutions were then spotted on drug-free agar plates. Plates
were
incubated at 37°C for 1 day (
S. aureus) or 3 days
(
M. smegmatis),
and the colonies were counted. Bactericidal
activity was expressed
as percent survival relative to the CFU per
milliliter at the
time of fluoroquinolone addition. The 90% lethal
dose (LD
90) was
the fluoroquinolone concentration at which
survival was 10% that
observed with an untreated control plated at the
time of fluoroquinolone
addition.
Selection of third-step mutants of S. aureus.
S. aureus strain EN1252 was grown to stationary phase by
overnight incubation in CY medium, the cells were harvested by
centrifugation (4,000 × g for 10 min), and they were
then resuspended in CY medium at a dilution of 1:10 relative to the
culture prior to centrifugation. Fluoroquinolones (see Table 3) were
added at lethal concentrations (fourfold the MIC99 for each
fluoroquinolone; see reference 5) to cell suspensions to
enrich third-step mutant fractions of the populations. Incubation was
for 5 h, after which cells were again concentrated by
centrifugation. Cells (5 × 107 CFU/ml) were then
applied to agar plates containing the same fluoroquinolone used for
enrichment of mutants at concentrations of two and threefold the
MIC99. Colonies were recovered after incubation at 37°C
for 2 to 3 days. Cells from colonies were spread on drug-free GL agar
to obtain single colonies after incubation for 1 day at 37°C;
subsequent testing on quinolone-containing GL agar confirmed that the
putative third-step mutants were capable of growth on the selective
fluoroquinolone at the selection concentration.
DNA sequence determination.
The nucleotide sequences of the
quinolone-resistance-determining region (QRDR) of gyrA, parC,
gyrB, and parE were determined after amplification of
the respective DNA fragments from S. aureus chromosomal DNA
templates using PCR. Cells in 1 ml of late-log-phase culture were
harvested by centrifugation and resuspended in 100 µl of SST buffer
(50 mM NaCl, 20% sucrose [wt/vol], 50 mM Tris-HCl [pH 7.6]).
Lysostaphin (AMBI Inc., Purchase, N.Y.) and RNase A (Sigma Chemical
Corp., St. Louis, Mo.) were added to final concentrations of 100 and
200 µg/ml, respectively. After incubation at 37°C for 10 min
followed by 3 min on ice, samples were mixed with 300 µl of general
lysis buffer (100 mM NaCl, 10 mM Tris-HCl [pH 7.9], 1 mM EDTA [pH
8.0]) plus 20 µl of 20% Triton X-100. Samples were then boiled for
10 min. Cell debris was removed by centrifugation, and 8 µl of
DNA-containing supernatant fluid was used for each 100-µl PCR.
PCRs involved incubations at 95°C for 2 min followed by 30 cycles at
93°C for 1 min 20 s, 49°C for 1 min, and 72°C for 1 min.
After each cycle the 72°C elongation phase was extended by 1 s.
The PCR products were purified with a Qiagen PCR purification
kit, and
nucleotide sequences were determined in the forward direction
with an
automated DNA sequencer. Primers SA-
parC.seq (5' ACG
TCG
TAT TTT ATA TGC AA-3'), SA-
gyrAfwd
(5'-AGA TTA TGC GAT GAG TGT
TAT CGT TGC-3'), SA
parE.seq (5'-AAA AAG CGA TTA AAG CAC AAC AAG
CAA-3'),
and SA
gyrB.seq (5'-GTC GCA CGT ACA GTG GTT GAA
AAA GG-3')
were used for sequencing after PCR amplification of
DNA fragments
using primers SA-
parCfwd (5'
TGA TGA GGA GGA AAT CTA GTG-3'),
SA-
parCrev (5' GGA AAT CTT GAT GGC
AAT AC-3'), SA-
gyrAfwd (defined
above),
SA-
gyrArev (5'-TAG TCA TAC GCG CTT CAG TAT
AAC GCA-3'),
SA-
parEfwd (5'-CAA ACG
AAA TCT AAA TTG GGT ACT TCT-3'),
SA-
parErev (5'-TCT TCG TCT GTC CAA
GCG TAT T-3'), SA-
gyrBfwd (5-TGG TAC
GCA
TGA AGA CGG ATT C-3'), and SA-
gyrBrev
(5'-GAC CTT TGT ATA GCG
CAA TAG ACC ATT-3').
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RESULTS |
Effect of C-8 substituents on bacteriostatic activity.
We have
argued that inhibition of growth by quinolones occurs at lower drug
concentrations than cell death when the latter is measured using short
incubation times (5, 31). To better distinguish between
the two processes and to attain greater accuracy, we measured
bacteriostatic activity by determining the MIC99 rather than the more standard inhibition of 99.99 to 99.999%
(23). MIC99s for wild-type M. smegmatis are listed in Table 1,
where the fluoroquinolones are grouped according to their C-7
ring structure (Fig. 1 shows structures
of compounds). With M. smegmatis the C-8 halogen and methoxy
groups improved the ability to block growth by about threefold when the
compounds had small C-7 rings. The larger C-7 ring systems present on
PD158804 and Bay y-3114 rendered these C-8 hydrogen compounds
more active than their C-8-modified derivatives. With the gyrase
mutant, however, all of the C-8-modified compounds were more active
than their C-8 hydrogen derivatives (Table 1). Thus, a C-8-halogen or
methoxy group increases bacteriostatic activity against a resistant
variant of M. smegmatis in which glycine has been
substituted for aspartic acid at GyrA position 95.
We next examined
S. aureus as an example of the more complex
situation in which two mutant quinolone targets are present.
In this
mutant, position 84 is Leu in GyrA (gyrase) and position
80 is Phe in
ParC (also known as GrlA, a subunit of topoisomerase
IV; we use
the ParC designation below to make
S. aureus nomenclature
consistent with that of other bacteria). The general pattern of
susceptibility was similar to that observed with
M. smegmatis (Table
1), supporting the conclusions drawn above.
However, the
enhancing effect of the moieties attached to the C-8
position
was less with wild-type
S. aureus than with
wild-type
M. smegmatis and more with mutant
S. aureus than with mutant
M. smegmatis (Table
1). These
differences between the two organisms were expected
because wild-type
M. smegmatis, like many other mycobacterial
species, behaves
as though it is a moderately resistant mutant
(
14,
15).
With mutants of both species enhancement was usually
least for a C-8
fluorine group and most for the C-8 chlorine and
C-8 methoxy
substituents.
An appropriate C-8 hydrogen compound was unavailable for directly
evaluating the effect of the C-8 fluorine in sparfloxacin
(sparfloxacin
contains a C-5 amino group absent from the other
compounds tested).
However, the closely related C-8 fluorine derivative,
PD125232, was
more active against mutants than its C-8-hydrogen
cognate PD158804
(Table
1). Thus, the C-8 fluorine of sparfloxacin
probably contributes
to activity against
mutants.
Effect of C-8 substituents on bactericidal activity.
Lethal
activity, when measured with short incubation times, probably arises
from the formation of quinolone-topoisomerase-DNA complexes followed by
release of double-strand DNA breaks from the complexes
(5). To assess the action of C-8 substituents on the
combined effect of these two events, cultures of M. smegmatis and S. aureus were incubated with each of the
12 fluoroquinolones. Then, the fraction of surviving cells was
determined (see Materials and Methods). Plots of survival at various
quinolone concentrations show that few of the C-8-substituted compounds
were more lethal than their C-8 hydrogen derivatives against wild-type
M. smegmatis (Fig. 2) and
S. aureus (Fig. 3). The effect
of the C-8 moieties on lethal action was more pronounced with the
resistant GyrA variant of M. smegmatis (Fig.
4) and with the GyrA ParC variant of
S. aureus (Fig. 5) than with
wild-type strains, as shown by LD90 determinations (Table
2). Against the mutants the C-8 bromine and methoxy groups conferred the most activity; in some cases a
C-8-fluorine moiety improved activity, while in others it did not.
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FIG. 2.
Bactericidal activities of fluoroquinolones against a
wild-type strain of M. smegmatis. M. smegmatis
strain mc2155 was grown to mid-log phase and then treated
with the indicated concentration of fluoroquinolone for 18 h.
Dilutions were prepared, and aliquots were applied to drug-free agar
plates. After incubation for 2 to 3 days, colonies were counted, and
the fraction of surviving cells was calculated. (A) Ciprofloxacin
(C-8-H, open circles) and PD129603 (C-8-C1, filled circles). (B)
PD138032 (C-8-H, open circles), PD125275 (C-8-F, filled diamonds), PD
163753 (C-8-Br, filled squares), PD138124 (C-8-Cl, filled triangles),
and PD135432 (C-8-methoxy, filled circles). (C) PD158804 (C-8-H, open
circles), sparfloxacin (C-8-F, filled circles), and PD125232 (C-8-F,
filled squares). (D) Bay y-3114 (C-8-H, open circles) and moxifloxacin
(C-8-methoxy, closed circles). Similar results were obtained in a
replicate experiment.
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FIG. 3.
Bactericidal activities of fluoroquinolones against a
wild-type strain of S. aureus. Strain ISP794 was grown to
mid-log phase and then treated with the indicated concentration of
fluoroquinolone for 2 h. Dilutions were prepared and aliquots were
applied to drug-free agar plates. After incubation for 1 day, colonies
were counted and the fraction of surviving cells was calculated. (A)
Ciprofloxacin (C-8-H, open circles) and PD129603 (C-8-Cl, filled
circles). (B) PD138032 (C-8-H, open circles), PD125275 (C-8-F, filled
diamonds), PD 163753 (C-8-Br, filled squares), PD138124 (C-8-C1, filled
triangles), and PD135432 (C-8-OMe, filled circles). (C) PD158804
(C-8-H, open circles), sparfloxacin (C-8-F, filled circles), and
PD125232 (C-8-F, filled squares). (D) Bay y-3114 (C-8-H, open circles)
and moxifloxacin (C-8-methoxy, closed circles). Similar results were
obtained in a replicate experiment.
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FIG. 4.
Bactericidal activities of fluoroquinolones against a
gyrA resistance mutant of M. smegmatis. M. smegmatis strain KD2003 was grown to mid-log phase and then
treated with the indicated concentration of fluoroquinolone for 18 h. Dilutions were prepared, and aliquots were applied to drug-free agar
plates. After incubation for 2 to 3 days, colonies were counted, and
the fraction of surviving cells was calculated. Panels and symbols are
the same as those in Fig. 2. Similar results were obtained in a
replicate experiment.
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FIG. 5.
Bactericidal activities of fluoroquinolones against a
parC gyrA resistance mutant of S. aureus. Strain
EN1252 was grown to mid-log phase and then treated with the indicated
concentration of fluoroquinolone for 2 h. Dilutions were prepared
and aliquots were applied to drug-free agar plates. After incubation
for 1 day, colonies were counted and the fraction of surviving cells
was calculated. Symbols and panels are the same as those in Fig. 3.
Similar results were obtained in a replicate experiment.
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Determination of fluoroquinolone target in S. aureus.
Since S. aureus has two topoisomerase
targets for the fluoroquinolones, we did not know a priori whether the
data described above for the mutant reflected activity against
resistant gyrase or resistant topoisomerase IV. Since the more
susceptible target is expected to be under more selective pressure than
the less susceptible one, the identity of a third-step mutation should reveal the principle target in the double mutant. We obtained third-step (triple) mutants from the double mutant by selection with
six of the fluoroquinolones and then determined the nucleotide sequences of the QRDR (28) of gyrA and
parC. Most of the compounds selected GyrA variants in which
serine-85 was changed to proline (Table
3). We were able to use levofloxacin, a
fluoroquinolone whose structure is distantly related to that of
compounds in the present set, to rule out the possibility that
third-step ParC variants were inviable in the genetic background
tested: of three independent mutants selected by levofloxacin, two were
variants of ParC; one lacked a mutation in the QRDR of either
gyrA or parC (data not shown). Taken together the
data indicate that resistant gyrase was the target in the S. aureus mutant for the compounds listed in Table 3.
Several additional features of Table
3 require comment. First, the
propensity for selection of the Ser-85-to-Pro variant,
rather than a
more common position 88 variant, may be related
to the preexisting
amino acid change at position 84. A comparable
change occurred when the
reverse experiment was performed with
Escherichia coli:
Ser-83 (equivalent to position 84 in
S. aureus)-to-Leu
variants were selected when the parental strain contained a
Ser-84-to-Pro
change (data not shown). Rules governing selection of
multiple
changes in gyrase are under investigation. Second, the C-8
chlorine
derivative PD138124 selected both GyrA and ParC variants. With
this compound some cells in the population appear to have mutant
gyrase
as the primary target while other cells have mutant topoisomerase
IV as
the main target. Apparently a change in either protein lowers
susceptibility. Third, the QRDRs for three mutants lacked nucleotide
sequence changes in either
gyrA or
parC. For
these cases additional
work is required to determine whether resistance
is due to nontarget
mutations, as reported previously for second-step
mutants (
11),
or to amino acid changes in GyrA that are
outside the QRDR (
16).
Changes in the GyrB or ParE
proteins are not likely explanations
because nucleotide sequence
analysis indicated that the QRDRs
of these two proteins were identical
in the three mutants to those
of the parental strain (data not
shown).
| |
DISCUSSION |
The experiments described above indicate that C-8 fluorine,
chlorine, bromine, and methoxy moieties increase the activity of
fluoroquinolones against fluoroquinolone-resistant mutants of M. smegmatis and S. aureus. The potency measures used in
the present work (MIC99 and LD90) differ from
those used by clinical laboratories (MIC and minimum bactericidal
concentration). However, the rank order of the compounds is likely to
be similar; thus, we expect the activity increase associated with C-8
halogen or methoxy groups to be observed when NCCLS assays are employed.
Measurement of MIC99 with S. aureus (Table 1)
showed that C-8 substituents enhance the ability to inhibit growth of
mutants more than growth of wild-type cells. This phenomenon, which has also been observed for a C-8 methoxy compound with E. coli
(31), was not as obvious with M. smegmatis
(Table 1). Since wild-type gyrase of M. smegmatis has an
amino acid sequence and behavior similar to first-step mutants of other
bacteria (14, 15), the similarity between wild-type and
mutant M. smegmatis is consistent with a preferential
enhancement of activity with mutants.
Biochemical experiments with an engineered, quinolone-resistant gyrase
of E. coli support the assumption that the differences among
the compounds reflect effects on topoisomerases. Purified, resistant
enzyme showed greater quinolone susceptibility, relative to wild-type
enzyme, according to the following order (from greatest to least
activity): C-8 bromine and C-8 chlorine, C-8 fluorine and C-8
ethoxy, and C-8 hydrogen (2). Our intracellular data (Table 1) showed a similar series, although we examined C-8 methoxy rather than C-8 ethoxy effects. The methoxy group was generally among
the most active. Preferential improvement of activity against resistant
mutants can be explained if the recognition helix of the GyrA protein
(22) serves as an important portion of the quinolone
binding site (21). Resistance mutations that lower susceptibility the most tend to reduce the electron-rich character of
the recognition helix. The C-8 halogen and C-8 methoxy fluoroquinolone substituents may partially restore that microenvironment, thereby improving drug binding. Although it is likely that resistant gyrase was
the primary target for M. smegmatis (the related M. tuberculosis lacks genes encoding topoisomerase IV
[7]) and in most cases for S. aureus (Table
3), the principle sketched above could also apply to topoisomerase IV
(16).
Enhanced lethal activity, which was most noticeable with the mutants
(Fig. 2 through 5; Table 2), is probably due in part to increased
ability to form ternary complexes (lower MIC99s; Table 1).
We have argued previously that C-8 moieties confer an additional
activity against mutants that is likely to involve the release of
breaks from the complexes (8, 31). Determination of the
LD90, which reflects both events, shows that C-8 halogen and methoxy groups increase lethal activity, although with some compounds the C-8 fluorine has little effect (Table 2).
We conclude that the presence of a C-8 fluorine, chlorine, bromine, or
methoxy moiety helps define a set of fluoroquinolones that is
distinguished by increased activity against resistant gyrase mutants.
In addition to findings with M. smegmatis and S. aureus, described above and in the references (17, 26, 30), selected representatives of the C-8-substituted group have shown increased activity with mutants of E. coli (21,
31), Pseudomonas aeruginosa (20),
Mycobacterium bovis BCG (8, 29), and M. tuberculosis (8, 29). Thus, it is likely that the
properties of the group apply to many bacterial species. Since some
members of the group, such as sparfloxacin, gatifloxacin, and
moxifloxacin, are being used clinically, it may be possible to test the
idea (10) that these compounds will cause resistance to
develop more slowly than a C-8 hydrogen fluoroquinolone such as ciprofloxacin.
| |
ACKNOWLEDGMENTS |
We thank Jerome Schentag for suggesting a study of quinolone
relationships and the following for critical comments on the manuscript: Marila Gennaro, Samuel Kayman, and Anthony Maxwell. We also
thank Anthony Maxwell for communicating unpublished observations.
This work was supported by grants from the NIH (AI35257), Mylan
Pharmaceutical Co., and Bayer AG.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Public Health
Research Institute, 455 First Ave., New York, NY 10016. Phone: (212) 578-0830. Fax: (212) 578-0804. E-mail:drlica{at}phri.nyu.edu.
| |
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Antimicrobial Agents and Chemotherapy, October 2001, p. 2703-2709, Vol. 45, No. 10
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.10.2703-2709.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
