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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, December 2000, p. 3337-3343, Vol. 44, No. 12
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mutant Prevention Concentration as a Measure of
Fluoroquinolone Potency against Mycobacteria
Georg
Sindelar,1
Xilin
Zhao,1
Anthony
Liew,1
Yuzhi
Dong,1
Tao
Lu,1
Jianfeng
Zhou,1
John
Domagala,2 and
Karl
Drlica1,*
Public Health Research Institute, New York,
New York 10016,1 and Parke-Davis
Pharmaceutical Research Division, Warner Lambert Company, Ann
Arbor, Michigan 481052
Received 17 April 2000/Returned for modification 24 July
2000/Accepted 11 September 2000
 |
ABSTRACT |
Mutant prevention concentration (MPC) has been proposed as a new
measure of antibiotic potency by which the ability to restrict selection of resistant mutants is evaluated. To determine whether MPC
provides potency information unavailable from the more customary measurement of the MIC, 18 fluoroquinolones were examined for their
ability to block the growth of Mycobacterium smegmatis and to select resistant mutants from wild-type populations. Both MPC and
MIC were affected by changes in the moiety at the fluoroquinolone C-8
position and in alkyl groups attached to the C-7 piperazinyl ring. When
eight resistant mutants, altered in the gyrase A protein, were tested
with fluoroquinolones having either a methoxy or a hydrogen at the C-8
position, the MIC for the most resistant mutant correlated better with
the MPC than did the MIC for wild-type cells. For C-8-fluorine
derivatives, which were generally less active than the C-8-methoxy
compounds but which were more active than C-8-hydrogen derivatives, the
MICs for both the mutant and the wild type correlated well with the
MPCs. Thus, measurement of the MICs for wild-type cells can reflect the
ability of a quinolone to restrict the selection of resistance, but
often it does not. With the present series of compounds, the most
potent contained a C-8-methoxy and a small group attached to the C-7 ring.
| |
INTRODUCTION |
To develop more effective
antituberculosis agents, we have been studying the fluoroquinolones.
These compounds, which act by trapping gyrase on DNA, are
generally used only as second-line therapeutics because
resistance mutations in Mycobacterium tuberculosis often
render them ineffective. For example, in the early 1990s a strain of
M. tuberculosis resistant to isoniazid, rifampin, streptomycin, and ethambutol spread among immunocompromised persons in
New York City (1). Patients were treated with the
fluoroquinolone ciprofloxacin, and within a few months some were
found harboring strains of M. tuberculosis that were
resistant to fluoroquinolones as well as to the four other agents
(11). When we examined these strains, we noticed that some
fluoroquinolones (e.g., sparfloxacin) were more effective than others
(e.g., ciprofloxacin) at blocking the growth of resistant mutants after
the data were normalized to the results obtained with wild-type cells
(5, 12). This observation led to the idea that some
quinolones might be better than others at trapping resistant gyrase in
mycobacteria. Subsequent work showed that compounds with a methoxy
group attached to the C-8 position (C-8-OMe) were particularly
effective against resistant mutants (5, 13).
While examining the effects of C-8-OMe groups we observed a complex
relationship between the recovery of resistant mutants and the
fluoroquinolone concentration (6). Increases in
fluoroquinolone concentration cause the fraction of cells that form
colonies on quinolone-containing agar to drop sharply to a plateau and
then drop sharply a second time. We proposed that the plateau is due to
the presence of first-step, resistant mutants in the population. If
this were true, the second drop in mutant recovery should occur when
the MIC for these mutants is approached: once the growth of first-step
mutants is blocked, no mutant should be recovered at the cell amounts
used because a rare, double mutation would be required to overcome the
quinolone effect. The minimal quinolone concentration that allows no
mutant recovery when more than 1010 cells are applied to
drug-containing agar was defined as the mutant prevention concentration
(MPC). If relevant concentrations of an antibiotic in tissue can be
maintained above the MPC, selection of resistant mutants should be
severely restricted.
MPC has been measured for only a few fluoroquinolones;
consequently, structure-activity relationships are still poorly
defined. In the present work we determined the MICs and MPCs for
Mycobacterium smegmatis of fluoroquinolones that differed in
moieties attached to the C-8 position and to the C-7 piperazinyl ring.
C-8 substituents influenced the effect that C-7-ring alkyl groups have
on fluoroquinolone MICs and MPCs. When resistant gyrA
(gyrase) mutants were examined, fluoroquinolone susceptibility
varied with drug structure in a way that allowed inferences about
quinolone binding to gyrase. Data obtained with the mutants also
revealed that the MPC often correlates better with the MIC for the most
resistant first-step mutant than with the MIC for wild-type
cells, consistent with the two-mutation explanation for MPC.
Collectively, these data show that the MPCs for and the
susceptibilities of resistant mutants can be used to discriminate among
closely related fluoroquinolones.
| |
MATERIALS AND METHODS |
Bacterial strains and growth.
The relevant features of
M. smegmatis mc2155 and its gyrA
mutants are listed in Table 1. The cells
were grown as liquid cultures in Middlebrook 7H9 medium containing 10%
albumin dextrose complex and 0.05% Tween 80 (8). Colonies
were grown on Middlebrook 7H10 agar plates containing a
fluoroquinolone. The incubation temperature for growth was 37°C.
The MIC was determined graphically from log-log plots of the
fluoroquinolone concentration on agar plates versus the fraction of CFU
recovered, and it was defined as the concentration required to inhibit
colony formation by 99% (MIC99) after incubation for 3 days. Cultures contained about 5 × 108 CFU per ml
prior to plating. For these determinations, fluoroquinolone concentration increments differed by 0.01 µg/ml for wild-type cells
and by 0.1 or 0.5 µg/ml for resistant mutants, depending on the
compound and the mutant. To assay the selection of
fluoroquinolone-resistant mutants, M. smegmatis was grown to
the stationary phase (350 Klett units), harvested by centrifugation
(6,000 × g for 15 min), and resuspended in the same
volume of fresh growth medium. Incubation was continued until the
turbidity of the culture reached 400 to 450 Klett units, and the
culture was again concentrated by centrifugation. The cells were
resuspended to about 109 CFU per ml in growth medium and
were then applied at various volumes and concentrations to
fluoroquinolone-containing 7H10 agar at a maximum of 2 × 109 CFU per 100-mm-diameter agar plate. The plates were
allowed to dry in a biosafety cabinet for about 30 min at room
temperature, sealed with electrical tape to minimize evaporation, and
inverted for incubation at 37°C for 3 to 7 days. After the colonies
were counted, they were confirmed to be composed of resistant mutants by regrowth on fluoroquinolone-containing agar.
Fluoroquinolones.
Several sets of fluoroquinolones were
provided by Parke-Davis Pharmaceutical Company, Ann Arbor, Mich. (for
structures, see Fig. 1). PD135432 has the
same structure as AM1155 (gatifloxacin). Ciprofloxacin was obtained
from Miles Laboratories (West Haven, Conn.). The fluoroquinolones (10 mg) were dissolved in 0.1 ml of 1 N NaOH (1/10 of the final volume),
and then sterile water was added to give a final concentration of 10 mg/ml. This stock solution was divided into 50-µl aliquots and was
stored at 
80°C. Dilution series were prepared with autoclaved
water. Solutions were occasionally stored at 20°C for several
weeks.
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FIG. 1.
Fluoroquinolone structures. A structure common to the
compounds tested is shown, with the variable groups marked X, Y, Z, and
C8. Identification numbers for the compounds are listed below the
structures. Due to rotation at the C-7 position, substitutions at the
3' (X) and 5' (Z) positions are identical.
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RESULTS AND DISCUSSION |
Effects of C-8- and C-7-ring substituents on inhibition of
wild-type growth by fluoroquinolones.
The bacteriostatic effects
of substituents attached to the C-8 position and to the C-7 piperazinyl
ring of N-1-cyclopropyl fluoroquinolones were examined by
measuring the MIC99 for wild-type M. smegmatis.
Three sets of compounds differing at the C-8 position (C-8-H, C-8-OMe,
and C-8-F) were compared; within each set, the compounds differed in
substituents attached to the C-7 piperazinyl ring (Fig. 1). With the
C-8-H series, optimal activity was observed when two methyl groups were
attached to the C-7 ring (Table 2, strain
KD1163), as reported previously for Mycobacterium avium (9). For the C-8-OMe compounds, several C-7-ring
substituents (dimethyl, monomethyl, N-ethyl, and
C-3'-ethyl) improved the bacteriostatic activity by three-
to fourfold, while the N-isopropyl substituent had no effect
(Table 2, strain KD1163). Each member of the C-8-OMe set had a lower
MIC99 than its counterpart in the C-8-H set. The activity
of the most potent C-8-F compound, in terms of absolute MIC99, was in the same range as that of the most active
C-8-OMe compounds. However, the C-8-F series exhibited a pattern
different from that seen with the two other sets in that the compound
lacking a C-7-ring alkyl was quite active. As the size of the alkyl
groups increased beyond the monomethyl level, the activities of the
C-8-F derivatives decreased. Collectively, these data establish that the nature of the C-8 moiety influences the bacteriostatic effect of
C-7-ring alkyl groups. Additional experiments are now required to
attribute specific structures to effects on drug uptake, efflux, and
trapping of gyrase on DNA.
Since the optimal C-7-ring substituents were one and two methyl groups,
we added C-8-Cl and C-8-Br fluoroquinolones to the series to assess the
effect of altering the C-8 moiety. For the monomethyl series,
compounds containing halogens or a C-8-OMe group exhibited
similar effectiveness, which was about threefold better than
that of the compound lacking a C-8 substituent (Table 2, strain
KD1163). In the case of the dimethyl series, the C-8-OMe derivative was
the most active; compounds with C-8 halogens varied considerably
in activity, and the compound lacking a C-8 group was as
effective as some halogenated compounds. Collectively, these
measurements indicate that fluoroquinolones with a C-8-OMe group are
generally more effective against wild-type M. smegmatis than
compounds with fluorine or hydrogen at position C-8 and that they are
most potent when a small alkyl group is attached to the C-7 ring.
Effect of C-8- and C-7-ring substituents on inhibition of growth of
first-step fluoroquinolone-resistant gyrase mutants.
The
relationship between fluoroquinolone structure and activity against
resistant gyrA mutants was examined by determining the
MIC99s of the C-8-OMe, C-8-H, and C-8-F
fluoroquinolones for eight mutants of M. smegmatis
(Table 2, strains labeled as GyrA amino acid changes). The C-8-OMe
compounds were the most effective, followed by the C-8-F derivatives
(see averages for GyrA variants at the bottom of Table 2). Within the
C-8-OMe series, the C-7-ring monomethyl derivative was generally the
most effective; for both the C-8-F and C-8-H series, the monomethyl
compound was optimal about as often as the dimethyl derivative was.
Compounds with large alkyl groups on the C-7 ring were generally least effective.
The GyrA G89C variant and several substitutions at position 95 were
associated with greater resistance than that for the other variants
examined (summarized in Table 3). A
comparison of two C-7-ring ethyl compounds showed that the position of
the C-7-ring alkyl affects the identity and susceptibility of the most
resistant variant, particularly when there is a methoxy or fluorine
attached to the C-8 position: for the N-ethyl compounds, the
G89C amino acid change was associated with the most resistance; for the
C-3'-ethyl derivatives, the D95 substitutions were
associated with resistance similar to or greater than that for the G89C
alteration (Table 3). When the mutants were compared for the effects of
the position of the piperazinyl ring ethyl by determination of the MIC
ratio (Table 4), the G89C variant was
distinctly less susceptible than the A91V and D95 mutants to the
N-linked ethyl compounds, at least when a methoxy or fluorine
substituent was attached to the C-8 position. In other work we found
that GyrA G89C variants are preferentially selected by high
concentrations of the C-7-ring N-ethyl compound, while D95G
variants are selected by the C-3'-ethyl derivative (14), consistent with the data in Tables 3 and 4. The
N-isopropyl group also preferentially lowered the activity
against the G89C GyrA variant (Table 4). If we assume that comparisons
among gyrase mutants reflect differences in intracellular quinolone
binding to gyrase, these data indicate that N-linked alkyl groups
interfere with fluoroquinolone binding to a GyrA protein that contains
a Cys-89 mutation, particularly when a methoxy or halogen is
attached to the C-8 position.
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TABLE 3.
Effect of fluoroquinolone C-8- and C-7-ring substituents
on the identities and susceptibilities of the most resistant
gyrA mutant of M. smegmatis
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Interference between Cys-89 and the N-alkylated C-7
piperazinyl ring can be explained by the fluoroquinolone
orientation on 
-helix 4 of the GyrA breakage-rejoining fragment.
From X-ray crystallography of the Escherichia coli GyrA
protein it has been argued that -helix 4 is located on the surface
of the protein, where it interacts with DNA (10). Amino acid
sequence similarities between the GyrA proteins of E. coli
and M. smegmatis indicate that amino acids 89, 91, and 95 of
the M. smegmatis GyrA protein reside in -helix
4. A negative interaction between Cys-89 and N-linked C-7-ring
alkyls suggests that the C-7-ring binds to gyrase near amino acid 89, while the remainder of the quinolone interacts with amino acids 91 and
95, the other two positions where strong resistance mutations map. Such
an alignment, which is shown schematically in Fig.
2, explains why a variant of E. coli with a GyrA G81D variation, which is equivalent to a position
89 mutation in M. smegmatis, exhibits resistance to
fluoroquinolones but not to nalidixic acid (3): nalidixic
acid lacks the C-7 ring required for interference.
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FIG. 2.
Orientations of fluoroquinolones and GyrA -helix 4. -helix 4, adapted from the crystal structure of the breakage-reunion
domain of the GyrA protein of E. coli (10), is
drawn parallel to the long axis of PD161144. Amino acid numbers
represent positions in the M. smegmatis GyrA protein.
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Effects of C-8- and C-7-ring substituents on selection of
resistant mutants.
When large numbers of cells, on the order of
108 to 1010, were incubated on agar
plates containing various fluoroquinolone concentrations, colony numbers dropped sharply in the presence of concentrations slightly above the MIC99. At higher concentrations a
plateau was seen for many compounds, followed by a second sharp drop at
very high concentrations (Fig. 3). To
assess the effect of C-7-ring substituents on the ability of a C-8-OMe
group to suppress selection of resistant mutants, four C-8-OMe
compounds that had similar MIC99s for wild-type cells were
compared (Fig. 3A). Differences among the four compounds were observed
with respect to the concentration at which the high-concentration drop
in recovery occurred: compounds with a C-3'-ethyl or
monomethyl substituent on the C-7 ring were the most effective,
followed by the dimethyl and N-ethyl compounds (Fig. 3A).
The compound with the lowest MIC99 for wild-type cells, the
C-7-ring dimethyl quinolone, ranked third when compared for the
concentration at which the second drop occurred. Thus, the MIC for
wild-type cells does not tightly correspond to the ability to suppress
selection of resistant mutants. The N-ethyl compound ranked
fourth, as expected, because the MIC99 of this compound for
the most resistant mutants was generally higher than those for the
other mutants examined (Table 3).
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FIG. 3.
Effect of small C-7-ring alkyls on selection of
resistant mutants by fluoroquinolones. M. smegmatis strain
KD1163 was applied to agar plates at the indicated fluoroquinolone
concentrations. Fluoroquinolones contained a C-8-OMe group (A), a C-8-H
group (B), and a C-8-F group (C). Symbols for panel A: squares,
PD138926, C-8-OMe C-7-ring dimethyl; diamonds, PD161144, C-8-OMe
C-7-ring N-ethyl; filled circles, PD161148, C-8-OMe C-7-ring
3'-ethyl; half-filled squares, PD135432, C-8-OMe C-7-ring methyl.
Symbols for panel B: squares, PD158804, C-8-H C-7-ring dimethyl;
diamonds, PD160788, C-8-H C-7-ring N-ethyl; filled circles,
PD160793, C-8-H C-7-ring 3'-ethyl; half-filled squares, PD138032, C-8-H
C-7-ring methyl. Symbols for panel C, squares, PD125232, C-8-F C-7-ring
dimethyl; half-filled squares, PD125275, C-8-F C-7-ring 3'-methyl;
diamonds, PD160791, C-8-F C-7-ring N-ethyl; filled circles,
PD160792, C-8-F C-7-ring 3'-ethyl. A replicate experiment focusing on
the second sharp drop in colony recovery gave results similar to those
shown.
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To determine whether the effects of the C-7-ring substituents for which
data are shown in Fig. 3A are influenced by the presence of the C-8-OMe
group, we examined a set of compounds that differed from the first
series in having either a C-8-H or a C-8-F moiety rather than a C-8-OMe
group. In the C-8-H series, the variation was wider for
MIC99 but narrower for the concentration at which the
high-concentration drop occurred (Fig. 3B). With the C-8-F series, the
MIC99 paralleled the concentration at the second sharp drop
in mutant recovery (Fig. 3C). Thus, the group at the C-8 position
appears to influence the relationship between wild-type MIC99 and the concentration needed to cause the second drop
in mutant recovery.
Two other C-8-OMe compounds, one with a C-7-ring
N-isopropyl group (PD162282) and one lacking a C-7-ring
substituent (PD135042), had MICs that were fivefold higher than those
discussed above (Table 2, strain KD1163). For this pair, the compound
lacking a C-7-ring alkyl substituent was remarkable for the absence of a plateau (Fig. 4A). The C-8-H and C-8-F
derivatives of this compound also exhibited little plateau regions
(Fig. 4B and C). The addition of an N-isopropyl group to the
C-7 ring lengthened the plateau for the C-8-OMe and C-8-H compounds
(Fig. 4A and B), consistent with the poor activity of the
N-isopropyl compounds against resistant mutants (Table 2).
For the C-8-F compounds, an isopropyl group attached to the C-7 ring
simply shifted the mutant recovery curve to higher concentrations (Fig.
4C). This behavior is currently unexplained. Collectively, the data in
Fig. 4 emphasize that the C-8 moiety influences the relationship
between MIC99 and the concentration associated with the
second drop in mutant recovery.
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FIG. 4.
Effect of C-7-ring isopropyl group on selection of
resistant mutants by fluoroquinolones. M. smegmatis was
applied to agar plates containing the indicated fluoroquinolone
concentrations. Fluoroquinolones contained a C-8-OMe group (A), a C-8-H
group (B), and a C-8-F group (C). Symbols for panel A: filled circles,
PD135042, C-8-OMe with no addition to C-7-ring; open circles, PD162282,
C-8-OMe C-7-ring N-isopropyl. Symbols for panel B: filled
circles, ciprofloxacin, C-8-H with no addition to the C-7 ring; open
circles, PD161645, C-8-H C-7-ring N-isopropyl. Symbols for
panel C: filled circles, PD117962, C-8-F with no addition to C-7 ring;
open circles, PD162281, C-8-F C-7-ring N-isopropyl. A
replicate experiment focusing on the second sharp drop in colony
recovery gave results similar to those shown.
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MPC.
For comparative purposes, we define MPC as the
concentration at which no colony is recovered when more than
1010 cells are applied to agar plates. MPC can be
determined by agar dilution, as is customary for measurement of MIC,
using concentration increments as fine as necessary. We prefer to
estimate MPC by interpolation, as shown in Fig.
5. The dashed line in Fig. 5 represents a
threshold below which more than 1010 cells must be applied
to agar plates to recover a single colony. The intersection of the
dashed line with the experimental data is used to estimate the MPC.
Table 5 shows the MPCs of the three sets
of fluoroquinolones by using the type of analysis used to obtain the
results presented in Fig. 5. The most potent compounds by this assay
were C-8-OMe derivatives with a C-7-ring methyl or ethyl group,
provided that the latter is attached to a ring carbon.
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FIG. 5.
Determination of MPC. Resistant M. smegmatis
mutants were recovered from agar plates containing the indicated
concentrations of PD161148 (C-8-OMe, C-7-ring 3'-ethyl) (A), PD160792
(C-8-F, C-7-ring 3'-ethyl) (B), and PD160793 (C-8-H, C-7-ring 3'-ethyl)
(C). MPC1010 is defined as the concentration at
which less than one colony is recovered when 1010 cells are
applied to agar plates. MPC1010 is approximated
by the intersection of the data curve and the dashed line in the
figure.
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A plot of the MPCs of the C-8-H and C-8-OMe compounds against the MICs
of each compound for the most resistant of the eight gyrA
mutants is shown in Fig. 6A. When we
assumed a linear relationship, the value of r (2)
was 0.92. Much more scatter was evident when the MPC was plotted
against the MIC99 for the wild-type cells (Fig. 6B). In
this case r was 0.39. For the C-8-F compounds, the MPCs
correlated well with the MICs for both mutant and wild-type cells (Fig.
6C and D). Thus, the MIC for the most resistant mutant correlates well
with the MPC; whether the MICs for wild-type cells also correlate with
the MPCs depends on the nature of the moiety at the C-8 position of the
fluoroquinolone. A correlation of the MPC with the MIC for the mutant
is consistent with the second drop in colony recovery on
quinolone-containing agar occurring at the point where the quinolone
concentration reaches the MIC for the most resistant mutant. At that
point resistance in a wild-type population would require two concurrent
mutations, which for M. smegmatis would occur at a frequency
of 10
14 to 1016 (single-mutation
frequencies were about 107 to 108; see the
plateau region in Fig. 3 and see reference 14). Such a
double-mutation frequency is orders of magnitude too low to be detected
by the methods used in the present work.
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FIG. 6.
Relationship between MPC and MIC. The MPC of each
fluoroquinolone, determined as described in the legend to Fig. 5, was
plotted against the MICs of C-8-OMe and C-8-H fluoroquinolones with the
C-7-ring moieties indicated in Fig. 2 for the most resistant
gyrA mutant (A) or wild-type cells (B). The relationships
between the MPC and the MIC for a gyrA mutant or the MIC for
the wild type are shown in panels C and D, respectively, for the C-8-F
compounds.
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Concluding remarks.
The present work expands the initial
description of MPC (6) by showing that differences can be
detected among a set of closely related fluoroquinolones and by showing
that the MPC correlates well with the MIC for the most resistant,
first-step gyrA mutant. Measurement of MIC and MPC also
revealed an unexplained interaction between C-8 substituents and
C-7-ring alkyl groups. To be clinically useful, the MPC must be below
the concentration in serum or tissue at the site of infection. Thus,
use of a combination of MPC and pharmacokinetic parameters provides a
way to compare antibacterial agents for their potential ability to
restrict the selection of resistant mutants. Of the first-line
antituberculosis agents tested, none has an MPC below the maximum
concentration in serum attained during therapy (7). However,
the MPCs of both PD135432 (gatifloxacin) and moxifloxacin, another
C-8-OMe fluoroquinolone, are below maximum concentration in serum
attained with doses recommended by the manufacturers (7).
Thus, administration of fluoroquinolones such that concentrations in
serum are above the MPC is feasible with existing compounds. Animal and
clinical studies are now needed to determine whether that will severely
restrict the selective enrichment of resistant mutants.
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ACKNOWLEDGMENTS |
We thank Marila Gennaro and Samuel Kayman for critical comments
on the manuscript.
This work was supported by NIH grant AI35257.
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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.
This is publication 74 from the Public Health Research Institute
Tuberculosis Center.
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Antimicrobial Agents and Chemotherapy, December 2000, p. 3337-3343, Vol. 44, No. 12
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
