Received 29 April 1998/Returned for modification 16 July
1998/Accepted 13 August 1998
Fluoroquinolones trap gyrase on DNA as bacteriostatic complexes
from which lethal DNA breaks are released. Substituents at the C-8
position increase activities of N-1-cyclopropyl
fluoroquinolones against several bacterial species. In the present
study, a C-8-methoxyl group improved bacteriostatic action against
gyrA (gyrase-resistant) strains of
Mycobacterium tuberculosis and M. bovis BCG. It
also enhanced lethal action against gyrase mutants of M. bovis BCG. When cultures of M. smegmatis, M. bovis BCG, and M. tuberculosis were
challenged with a C-8-methoxyl fluoroquinolone, no resistant mutant was recovered under conditions in which more
than 1,000 mutants were obtained with a C-8-H control. A
C-8-bromo substituent also increased bacteriostatic and lethal
activities against a gyrA mutant of M. bovis
BCG. When lethal activity was normalized to bacteriostatic activity,
the C-8-methoxyl compound was more bactericidal than its C-8-H control,
while the C-8-bromo fluoroquinolone was not. The C-8-methoxyl compound
was also found to be more effective than the C-8-bromo fluoroquinolone
at reducing selection of resistant mutants when each was compared to a
C-8-H control over a broad concentration range. These data indicate
that a C-8-methoxyl substituent, which facilitates attack of
first-step gyrase mutants, may help make fluoroquinolones effective
antituberculosis agents.
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INTRODUCTION |
As disease caused by the human
immunodeficiency virus spreads to regions of the world where infection
with Mycobacterium tuberculosis is common, the incidence of
active cases of tuberculosis is increasing dramatically due to weakened
host immunity (3, 22). The magnitude of this problem is
emphasized by the fact that more than one billion persons are already
infected with M. tuberculosis (22, 27). Effective
antituberculosis agents are available, but assuring patient compliance
to therapy regimens is expensive (5). Moreover, drug-resistant strains of M. tuberculosis, which cause
disease that is often very difficult to treat, occasionally arise and spread (1). These considerations suggest that tuberculosis will become an increasingly serious medical problem.
In an attempt to develop more effective antituberculosis agents, we
have been studying the fluoroquinolones. These compounds are generally
used only as second-line therapeutics because resistance of M. tuberculosis often renders them ineffective (for reviews, see
references 2 and 8). However,
studies with other bacteria have suggested ways to seek more effective
derivatives. For example, it has been proposed that two distinct events
occur when cells are treated with fluoroquinolones (7, 10).
The drugs first trap gyrase or topoisomerase IV on the chromosome as
fluoroquinolone-enzyme-DNA complexes in which the DNA is broken but
constrained by protein. These trapped complexes reversibly block DNA
synthesis and bacterial growth. In a second event, some of the
complexes release DNA ends, resulting in cell death. While inhibition
of growth is a convenient assay for drug potency, it sometimes fails to
identify the most lethal fluoroquinolone (29). Lethality is
likely to be important for minimizing the mutagenic action of
fluoroquinolones (20, 21) and the appearance of resistant
strains. Thus, fluoroquinolones should be evaluated on the basis of
lethal as well as bacteriostatic action. Another issue concerns the
finding that resistance to fluoroquinolones arises stepwise, usually
through mutations accumulating in the genes encoding DNA gyrase and DNA
topoisomerase IV (for reviews, see references 10 and
19). It has been possible to identify compounds that
have an increased ability to kill moderately resistant, first-step
mutants, and such compounds reduce the ability of resistant mutants to
be selected in wild-type populations (29). Those same
compounds may restrict the ability of mycobacteria to acquire
resistance mutations, since resistance in mycobacteria also arises
stepwise from mutations in gyrase (16, 26, 28).
In the present study we examined fluoroquinolone resistance and
intracellular activity in several species of Mycobacterium. With M. bovis BCG, both first- and second-step
mutations conferring resistance to ciprofloxacin appeared to be
located on alleles of gyrA, one of the genes encoding
gyrase. These strains, as well as wild-type M. smegmatis and
clinical isolates of M. tuberculosis, were used to study the
effects of fluoroquinolone structure. It had been suggested that C-8
alkoxyl groups might increase fluoroquinolone activity against M. avium (14), and both Pseudomonas aeruginosa and Staphylococcus aureus were known to be more susceptible
to C-8-modified fluoroquinolones (11, 13, 30). We
focused on a methoxyl (OMe) group at position C-8 because
fluoroquinolones having this substituent exhibit low mammalian
phototoxicity and only moderate cytotoxicity (18, 23, 24).
Against M. bovis BCG the C-8-OMe group increased both
bacteriostatic and lethal action, especially against first-step
gyrA resistant mutants. A C-8-bromo (C-8-Br) moiety was not
as active as the C-8-OMe group, particularly when lethal
action was normalized to bacteriostatic activity. These data
suggested that the C-8-OMe substituent may be especially effective at
facilitating the release of lethal DNA breaks from drug-gyrase-DNA
complexes. When wild-type cultures of all three species were tested for
the ability to acquire resistance mutations, fluoroquinolone
concentrations were found at which no mutant was recovered
following challenge with a C-8-OMe compound, while many were recovered
following challenge with a C-8-H control. Thus, a C-8-OMe substituent
makes fluoroquinolones more effective at attacking gyrase mutants
and at preventing selection of resistant mutants from wild-type
populations of mycobacteria.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
M.
smegmatis mc2155 was obtained from I. Smith, Public
Health Research Institute. M. bovis BCG (Bacille
Calmette-Guerin substrain Pasteur) was provided by Mounsef
Tiza, Public Health Research Institute, and was called KD1295. Strain
CX1 (9) was a derivative of KD1295 that contained a
gyrA mutation with a GAC to AAC change at codon 94, which
would change aspartic acid to asparagine; strain CX2 was a derivative
of CX1 that contained an additional gyrA mutation with a GCG
to GTG change at codon 90, changing alanine to valine. Clinical
isolates of M. tuberculosis were obtained from the Public
Health Research Institute Tuberculosis Center. All M. tuberculosis strains were identified by IS6110 DNA
typing as members of the W group, a set of clonal isolates recovered from persons affected during a recent outbreak of multidrug-resistant tuberculosis in New York City (4). Middlebrook 7H9 medium, enriched with 10% ADC (albumin-dextrose complex) and 0.05% Tween 80 (12), was used to grow mycobacteria in liquid culture.
Middlebrook 7H10 agar plates were used for single-colony isolation, MIC
determination, and measurement of CFU. Agar plates containing
fluoroquinolones were prepared by adding concentrated solutions to
molten agar. All experiments with M. tuberculosis were
carried out in a BSL3 containment facility.
Fluoroquinolones.
Ciprofloxacin was purchased from Miles
Pharmaceutical Company. Additional fluoroquinolones were provided by
Parke-Davis Pharmaceutical Company (see Fig. 1 for structures).
Fluoroquinolones were first dissolved in 1 N NaOH (1/10 final volume),
and then sterile water was added to yield a final concentration of 10 mg/ml. This stock solution was divided into 50-µl aliquots and stored
at 
80°C. Dilution series were prepared with sterile water.
Fluoroquinolones PD163753 and PD161144 correspond to compounds 3b and
5d, respectively, in a previous study (23).
Determination of mycobacterial sensitivity to
fluoroquinolones.
Bacteriostatic concentrations of the
fluoroquinolones were estimated in two ways. For growth in liquid
culture, cells were diluted in tubes containing various concentrations
of fluoroquinolone and then incubated until an untreated control
culture reached stationary phase (monitored by culture turbidity,
A600). The drug concentration required to
inhibit bacterial growth by 50% relative to the untreated control was
defined as the 50% inhibitory dose (ID50) (28).
The MIC was determined by plating dilutions of cultures on agar
containing various concentrations of fluoroquinolone. The concentration
that reduced the number of colonies by at least 99% relative to
untreated controls was taken as the MIC. Growth was at 37°C in the
presence of 5% CO2.
Bactericidal effects against M. bovis BCG were assessed by
determination of the number of CFU following fluoroquinolone treatment. Cultures were grown with horizontal rolling to mid-log phase
(approximately 3 × 108 cells/ml), and 1-ml aliquots
were transferred to sterile polystyrene tubes containing various
fluoroquinolones. After 48 h of incubation at 37°C, 100-µl
portions were diluted in 900 µl of fresh medium lacking drug. Serial
dilutions were made, aliquots were spread on 7H10 agar lacking drug,
and colonies were counted after 3 to 4 weeks' incubation.
Selection of resistant mutants.
Resistant mutants were
obtained by incubation of cultures spread on 7H10 agar plates
containing fluoroquinolone. A spontaneous gyrA
(Cipr) mutant, strain CX1, was obtained (9) by
growth of M. bovis BCG on agar containing 1 µg of
ciprofloxacin/ml. A culture of strain CX1 was then plated on agar
containing 10 µg of ciprofloxacin/ml to select a second-step
gyrA mutant, strain CX2. Nucleotide sequences in the
quinolone-resistance-encoding region of gyrA were determined by automated DNA sequencing following amplification by PCR
(26). For measurement of the ability of a fluoroquinolone to
reduce the selection of resistant mutants, mycobacterial cultures were grown to early stationary phase in 7H9 medium. For M. smegmatis, cells (1 ml) were then spread directly on 7H10
agar plates containing fluoroquinolone. For M. bovis BCG and
M. tuberculosis, cells (60 to 100 ml) were grown in roller
bottles, concentrated by centrifugation (3,000 × g for
10 min), resuspended in 20 ml of medium, and applied to agar plates in
1-ml aliquots at an estimated concentration of 2 × 109 to 30 × 109 CFU/ml. Colonies were
counted after incubation at 37°C for 7 to 10 days (M. smegmatis) or 21 to 28 days (M. bovis BCG and M. tuberculosis).
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RESULTS |
Bacteriostatic effects of fluoroquinolones.
Formation of
fluoroquinolone-gyrase-DNA complexes blocks bacterial growth;
consequently, the abilities of different fluoroquinolones to trap
gyrase on DNA can be compared by determining the drug concentration required to block colony formation or inhibit
growth in a liquid culture. In a preliminary search for
fluoroquinolones that are more effective than ciprofloxacin against
moderately resistant gyrase mutants of M. tuberculosis, we
compared the bacteriostatic activities of sparfloxacin and
ciprofloxacin using several clonal isolates of the multidrug-resistant
W IS6110 restriction fragment length polymorphism type
(28). Against a gyrA+ strain
(TN1626), sparfloxacin had the same effect as ciprofloxacin at one-half
the dose; against two resistant gyrA mutants, TN606 and
TN1625, only one-quarter the dose of sparfloxacin was required (Table
1). Thus, sparfloxacin was more effective
than ciprofloxacin at overcoming the protective action of a
gyrA mutation.
Since sparfloxacin and ciprofloxacin differ in several aspects
(structures are shown in Fig. 1), the
structural basis for the greater activity by sparfloxacin was not
clear. However, we suspected that the additional fluorine at position
C-8 in sparfloxacin was important, because substituents at this
position are known to increase the activity of fluoroquinolones against
other bacteria (11, 13, 23, 29, 30). To focus more
closely on C-8 substituents, we next compared two new compounds,
a C-8-OMe fluoroquinolone (PD161148) and its C-8-H control (PD160793),
for the ability to block growth of W type isolates of M. tuberculosis. These compounds and ciprofloxacin showed equal
ability to block growth of a gyrA+ strain
(TN1626) (Table 2); however, the C-8-OMe
compound inhibited growth at four-to-seven-times-lower concentration
than its C-8-H control or ciprofloxacin when gyrA mutants
were examined (Table 2). Thus, a C-8-OMe group increased the
bacteriostatic activities of fluoroquinolones against moderately
resistant, clinical isolates of M. tuberculosis.
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FIG. 1.
Structures of fluoroquinolones examined. The structure
common to the compounds is shown, with positions of variable groups
indicated by C5, C8, X, Y, or Z. Specific groups that distinguish the
compounds are shown at the bottom of the figure. Abbreviations: H,
hydrogen; Br, bromo; OMe, methoxyl; Me, methyl; Et, ethyl;
NH2, amino.
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A more extensive comparison of C-8 substituents was carried out with
strains of M. bovis BCG. Against the wild type, a C-8-OMe group doubled fluoroquinolone potency for growth inhibition while a
C-8-Br substituent had little effect (Table
3). Ciprofloxacin exhibited the least
activity. As expected, a gyrA (Cipr) mutation
(asparagine substituted for aspartic acid at codon 94)
increased the fluoroquinolone concentration required to inhibit growth
(strain CX1, Table 3). The ID50 ratio for first-step
mutant to wild type was 7- to 10-fold lower for compounds carrying
C-8-OMe and C-8-Br groups than for C-8-H derivatives (Table 3). Thus, C-8-OMe and C-8-Br groups increase the bacteriostatic action of fluoroquinolones against first-step gyrA resistant mutants
for slow-growing mycobacteria as they do for Escherichia
coli (reference 29 and data not shown). The
presence of an additional, second-step gyrA mutation, which
results in substitution of valine for alanine at codon 90 (strain CX2),
provided roughly the same additional (two- to fourfold) protection
against all of the compounds tested (Table 3). Thus, the enhancing
effect of C-8 substituents on bacteriostatic activity of
fluoroquinolones appears to be focused primarily on first-step mutants.
Bactericidal effects of fluoroquinolones.
Since
bactericidal action is not always predictable from
bacteriostatic activity (29), we measured the effects
of C-8 substituents on survival of M. bovis BCG
during fluoroquinolone treatment. Examination of Fig.
2 shows that a C-8-OMe group enhanced
fluoroquinolone lethality, particularly against a first-step
gyrA mutant. Similar data were obtained for another C-8-OMe
derivative, PD161144, when it was compared to the C-8-H control
compound PD160788 (data not shown).
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FIG. 2.
Effect of a C-8-OMe group on bactericidal action of
fluoroquinolones. (A) Wild-type M. bovis BCG (strain
KD1295) was incubated at the indicated concentrations of
fluoroquinolone for 48 h. Cultures were then diluted in drug-free
medium, and the number of viable colonies was determined by growth on
agar. Survival was calculated as the number of colonies recovered for
each fluoroquinolone concentration normalized to the number observed in
samples taken at the time of drug addition. (B) M. bovis BCG strain CX1 (first-step gyrA mutant) was
treated as described for panel A. (C) M. bovis BCG
strain CX2 (second-step gyrA mutant) was treated as
described for panel A. Arrows indicate concentrations that are 4 times
the ID50 for each compound. Symbols: circles, PD161148
(C-8-OMe); squares, PD160793 (C-8-H).
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We have argued that fluoroquinolones can be compared for their
abilities to stimulate release of DNA breaks from drug-gyrase-DNA complexes by normalizing lethal action to bacteriostatic effects (29). Since survival curves are complex, we have chosen to
compare the fractions of survivors for each compound at a concentration that is a fixed multiple of the bacteriostatic parameter,
ID50. In the present case, 4 times the ID50 was
used. Of wild-type cells, 4% survived treatment with PD161148
(C-8-OMe) at 4 times the ID50 (0.19 µg/ml), while 50%
survived incubation with its C-8-H derivative (4 times the
ID50, 0.32 µg/ml; arrows in Fig. 2A). With the first-step mutant (Fig. 2B), the difference in survival between the two compounds at 4 times the ID50 was about 60-fold. For the other
C-8-OMe/C-8-H pair (PD161144 and PD160788), the C-8-OMe substituent
increased lethal activity at 4 times the ID50 by about
5-fold for wild-type cells and about 50-fold for the first-step gyrase
mutant (strain CX1; data not shown). These data are consistent with the
C-8-OMe group enhancing the release of lethal DNA breaks, particularly in a gyrA mutant.
A C-8-Br moiety did not increase fluoroquinolone potency as much as a
C-8-OMe group. With wild-type M. bovis BCG, the
C-8-Br compound (PD163753) and its C-8-H control (PD138032)
exhibited similar dose responses for both bacteriostatic (Table 3) and bactericidal (Fig. 3) activity. Against
the first-step mutant CX1, the C-8-Br compound was more bacteriostatic
(Table 3) and allowed fewer survivors (Fig. 3) than its cognate C-8-H
derivative at a given concentration. However, the lethal actions of the
two compounds were equal at 4 times the ID50 (Fig. 3).
Thus, the enhancing effect of the C-8-Br substituent is exerted
largely at the level of bacteriostatic action, unlike the situation
described above for the C-8-OMe group. Nevertheless, the absolute
bacteriostatic and bactericidal activities of the C-8-OMe
(PD161148) and C-8-Br (PD163753) are similar (Table 2 and Fig. 2
and 3). This similarity probably arises from enhanced activity of the
C-8-Br compound due to the presence of a methyl group rather than an
ethyl group on its C-7-piperizinyl ring (see Fig. 1). The methyl
substituent improved both bacteriostatic and lethal activities,
as seen in comparisons of the C-8-H compounds PD160793 and
PD138032 (Table 3 and Fig. 2 and 3).
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FIG. 3.
Effect of a C-8-Br group on bactericidal action of
fluoroquinolones. M. bovis BCG strain KD1295 (wild
type; open symbols) or strain CX1 (first-step gyrA mutant;
filled symbols) was treated as in Fig. 2. Arrows indicate
concentrations that are 4 times the ID50 for each compound.
Symbols: circles, PD163753 (C-8-Br); squares, PD138032 (C-8-H).
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Based on other work it has been argued that two forms of lethal action
can be distinguished by using antagonists of RNA synthesis or protein
synthesis (7, 17). Inhibitors such as rifampin and
chloramphenicol block one pathway of fluoroquinolone lethality but not
the other (10). When chloramphenicol was tested for the
ability to protect M. bovis BCG from fluoroquinolone
action, the lethal action of the C-8-OMe compound PD161148 was shifted to higher concentrations, but the compound still killed M. bovis BCG (Fig. 4A). These data
indicate that two pathways of lethal action exist for C-8-OMe
fluoroquinolones and suggest that these agents may have
considerable activity even against nongrowing cells. Since a
similar protective effect was seen with the C-8-H control (Fig.
4B), activity in the presence of chloramphenicol is not unique to
fluoroquinolones containing a C-8-OMe group.
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FIG. 4.
Effect of chloramphenicol on bactericidal action of
C-8-OMe and C-8-H fluoroquinolones. (A) Wild-type M. bovis BCG was treated with the indicated concentrations of
PD161148 (C-8-OMe) in the presence (filled circles) or absence (open
circles) of 20 µg of chloramphenicol/ml added 60 min prior to the
fluoroquinolone. Incubation was continued for 48 h, after which
percent survival was determined as described in the legend for Fig. 2.
(B) Conditions were as described for panel A except that cells were
treated with PD160793 (C-8-H), in the presence (filled squares) or
absence (open squares) of chloramphenicol.
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Selection of fluoroquinolone resistance.
The data presented
above show that C-8 substituents make fluoroquinolones more
bacteriostatic and bactericidal against mycobacteria, especially when
the cells already contain fluoroquinolone-resistant gyrA
mutations. This raised the possibility that C-8 substituents would reduce the selection of resistance when wild-type cells were
challenged with moderate fluoroquinolone concentrations, since
the gyrA mutation plus a second mutation would be required for significant resistance. As a preliminary test of this idea, we
examined the rapidly growing species M. smegmatis by
plating cells on agar containing PD161148 (C-8-OMe) or its C-8-H
control, PD160793 (for this organism the two compounds inhibit growth
equally). As shown in Table 4, the
C-8-OMe group greatly reduced the selection of resistant mutants.
Restriction of mutant selection was not limited to C-8-OMe derivatives:
no mutants were obtained with the C-8-Br derivative under conditions in
which hundreds of mutants were obtained with its C-8-H control (data
not shown). The C-8-OMe group also reduced the selection of resistant
mutants in M. bovis BCG (Table 4). When the test was
applied to M. tuberculosis (clinical isolate TN1626),
no mutant was obtained with the C-8-OMe compound (PD161148) while more
than a thousand resistant mutants were recovered with the same
concentration of its C-8-H control (PD160793; Table 4). As expected,
the resistance mutations were mapped in gyrA (the nucleotide
sequences were determined for the regions of gyrA determining quinolone resistance in two M. tuberculosis
isolates following amplification by PCR, and in both cases a
substitution of glycine for aspartic acid was found at codon 94).
To assess the effect of drug concentration on mutant selection, we
determined the numbers of M. bovis BCG mutants that
arose on agar plates containing various fluoroquinolone concentrations (Fig. 5). The number of resistant mutants
declined as concentration increased, with the more potent compounds
generally allowing fewer mutations to arise at a given concentration.
Two features of Fig. 5 merit attention. First, the C-8-Br derivative
was more restrictive for mutant selection than its C-8-H control (Fig.
5), even though the two were equally potent against wild-type cells.
These two compounds differ in their activities against first-step
gyrA mutants (Table 3 and Fig. 3), and that is probably why
the C-8-Br compound allows fewer resistant colonies to arise. The
second noteworthy observation is that one C-8-H compound, PD138032, was
more effective than the other, PD160793, at reducing the selection of
resistant mutants (Fig. 5). This difference probably arises from
substituents on the C-7 ring, since they constitute the only difference
between PD138032 and PD160793 (Fig. 1).
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FIG. 5.
Effect of fluoroquinolone concentration on selection of
resistant mutants of M. bovis BCG. Wild-type cells were
spread on agar plates containing the indicated concentrations of
PD161148 (C-8-OMe; filled circles), PD160793 (C-8-H; filled squares),
PD163753 (C-8-Br; open circles), or PD138032 (C-8-H; open squares). The
plates were then incubated for 28 days at 37°C, and the number of
fluoroquinolone-resistant colonies was recorded.
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DISCUSSION |
C-8-OMe and C-8-Br substituents, when compared with
C-8-H controls, improved fluoroquinolone activity against
mycobacteria, particularly against moderately resistant gyrA
mutants. With respect to inhibition of growth, a C-8-OMe group had
little enhancing action against a wild-type isolate of M. tuberculosis, but the moiety made fluoroquinolones four- to
sevenfold more bacteriostatic against gyrA mutants (Tables 1
and 2). With M. bovis BCG, a C-8-OMe group doubled the
activity against wild-type cells and increased it by almost
10-fold against a first-step gyrA mutant (Table 3). A
C-8-Br group did not enhance bacteriostatic action with wild-type
cells, but it did with first-step mutants (Table 3). The C-8-OMe and
C-8-Br substituents probably increase the ability of fluoroquinolones
to block growth by facilitating formation of fluoroquinolone-gyrase-DNA
complexes (7). There is no evidence that complexes form with
topoisomerase IV in mycobacteria, since first-step and second-step
mutations map in the gyrase genes for both M. tuberculosis (16) and M. bovis BCG
(strains CX1 and CX2 of the present study). In this respect
mycobacteria may differ from bacteria such as E. coli,
S. aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria
gonorrhoeae, since parC (topoisomerase IV) mutants are
readily obtained in the latter organisms (reviewed in reference
10).
Enhancement of lethal action by a C-8-OMe group was readily observed
with wild-type and resistant mutants of M. bovis BCG (Fig. 2). Enhancement was probably due to an increase in the release of
double-stranded DNA breaks from drug-gyrase-DNA complexes
(7). Increased lability of the complexes was estimated
indirectly by determining percent survival at fluoroquinolone
concentrations normalized to correct for differences in growth
inhibition. For example, at 4 times the ID50, a C-8-OMe
group increased by 60-fold the fraction of cells killed for a
first-step gyrA mutant (Fig. 2). Apparently many more
drug-gyrase-DNA complexes form than release lethal double-stranded DNA
ends; a C-8-OMe group stimulates this release. Two forms of release can
be distinguished by sensitivity to chloramphenicol (7). The
activity enhancement due to a C-8-OMe group appears to affect both,
because chloramphenicol provided partial protection against both
C-8-OMe and C-8-H fluoroquinolones (Fig. 4).
The enhancing effect of the C-8-Br group, which is seen only with the
first-step gyrA mutant (Fig. 3), appears to differ
qualitatively from the effect of the C-8-OMe group: the fraction of
surviving cells is the same for C-8-Br and C-8-H compounds when the
data are normalized for bacteriostatic action (Fig. 3), while the
C-8-OMe compound is more lethal than its C-8-H control in this
comparison (Fig. 2). Lethality enhancement by the C-8-Br group probably
reflects an increase in trapping of gyrase-DNA complexes rather than in facilitated release of DNA breaks. Additional experiments are required
to determine whether alkyl groups on the C-7 ring, which differ in the
C-8-OMe and C-8-Br compounds, influence the action of C-8 moieties.
Since C-8 substituents facilitate attack of gyrA mutants, we
expected fewer resistant mutants to be recovered when cells were treated with C-8-OMe fluoroquinolones than with C-8-H derivatives. Two
tests were performed. In one, susceptible populations of M. smegmatis, M. bovis BCG, and M. tuberculosis were plated on agar containing a moderately high
concentration of fluoroquinolone (3.75 µg/ml). The presence of a
C-8-OMe group reduced the selection of resistant mutants (Table 4). For
the C-8-OMe compound this concentration was sixfold greater than the
ID50 of a gyrA mutant of M. bovis BCG, but for the C-8-H derivative it was only half the
ID50. Thus, there is a concentration window in which only the C-8-OMe compound prevents growth of mutants already present in the
susceptible population. The concentration window was clearly seen when
fluoroquinolone concentration was varied (Fig. 5).
The data shown in Fig. 5 also draw attention to the C-7 piperizinyl
ring: the C-8-H compound having a methyl group on its C-7 ring
(PD138032) was more effective than the compound having an ethyl group
(PD160793) (Fig. 5). Previous work indicates that alkylation of this
ring can influence bacteriostatic action against M. smegmatis (23) and M. avium
(15). We are now determining whether substituting a methyl
group for the ethyl group of PD161148 (C-8-OMe) will make a C-8-OMe
fluoroquinolone even more effective.
The activity enhancement by C-8 substituents, described above for
mycobacteria, is also observed with other bacteria. For example,
compounds containing a C-8-OMe group are more bactericidal against
quinolone-resistant S. aureus than are C-8-H derivatives, while C-8-Br and C-8-F derivatives are of intermediate potency (11, 30). A compound containing a C-8-Cl is more
bacteriostatic than its C-8-H derivative against quinolone-resistant
gyrA mutants of P. aeruginosa, a feature that is
also reflected in potency against gyrase purified from the strains
(13). With E. coli, a C-8-OMe group increases
fluoroquinolone lethality against gyrA mutants and reduces
the ability of the compound to select resistant mutants in wild-type
populations (29). Additional responses to quinolone
treatment shared by mycobacteria and other bacteria are rapid
inhibition of DNA synthesis (9, 25), fragmentation of DNA
(9, 25), and stepwise selection of resistance mutations in
the same regions of the gyrase genes (10, 16, 26) at about
the same frequency (10
8 to 109, data not
shown). Mycobacteria also display the unexplained phenomenon of
moderate concentrations of fluoroquinolone being more lethal than very
high ones (Fig. 2). This property is seen with many quinolones and many
bacterial species (reviewed in reference 10). Taken
together, these observations indicate that our general understanding of
fluoroquinolone action (10), obtained largely from
studies with E. coli, applies to mycobacteria.
A difference between gyrase from E. coli and that from many
mycobacteria is the alanine at position 90 of the mycobacterial GyrA
protein (6, 26). In E. coli and many other
bacteria, the equivalent position contains a serine or threonine, and
mutation of either to a hydrophobic amino acid is associated with
resistance. This may be why gyrase in many mycobacterial
species is moderately resistant to the fluoroquinolones (6).
The ease with which resistant M. tuberculosis strains
arise when patients are treated with compounds such as ciprofloxacin
may result from these agents blocking growth without effectively
killing cells. As a result, the mutagenic SOS response (20,
21) will be induced, and new gyrA mutants will add to
those already existing in the population. Many (80%) of the
gyrA mutations map at codon 94 (28), making the
cells effectively double mutants. If this idea is correct, a key
to developing more effective antituberculosis fluoroquinolones is
finding compounds that are so lethal that first-step mutants are
readily attacked and few wild-type cells survive to spawn such mutants.
Examination of C-8-Br and C-8-OMe derivatives for bacteriostatic
activity (Table 3), lethal action (Fig. 2 and 3), and reduction of
resistant mutant selection (Fig. 5) shows that adding C-8 substituents
to fluoroquinolones is a step in the right direction.
We thank S.-W. Lee and S. Moghazeh for technical assistance and
M. Gennaro, S. Kayman, B. Kreiswirth, T. Lu, and R. Pine for critical comments on the manuscript.
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Abstract
Introduction
