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Antimicrobial Agents and Chemotherapy, February 2001, p. 433-438, Vol. 45, No. 2
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.2.433-438.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mutant Prevention Concentrations of
Fluoroquinolones for Clinical Isolates of Streptococcus
pneumoniae
Joseph M.
Blondeau,1,*
Xilin
Zhao,2
Glen
Hansen,1 and
Karl
Drlica2
Departments of Clinical Microbiology, St.
Paul's Hospital (Grey Nuns') and Saskatoon District Health;
Department of Pathology, Royal University Hospital; and Department of
Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan,
Canada1; and Public Health Research
Institute, New York, New York 100162
Received 30 August 2000/Returned for modification 19 October
2000/Accepted 2 November 2000
 |
ABSTRACT |
The mutant prevention concentration (MPC) represents a threshold
above which the selective proliferation of resistant mutants is
expected to occur only rarely. A provisional MPC (MPCpr)
was defined and measured for five fluoroquinolones with clinical
isolates of Streptococcus pneumoniae. Based on their
potential for restricting the selection of resistant mutants, the five
fluoroquinolones, in descending order, were found to be
moxifloxacin > trovafloxacin > gatifloxacin > grepafloxacin > levofloxacin. For several compounds, 90% of
about 90 clinical isolates that lacked a known resistance mutation had
a value of MPCpr that was close to or below the serum levels that could be attained with a dosing regimen recommended by the
manufacturers. Since MPCpr overestimates MPC, these data identify moxifloxacin and gatifloxacin as good candidates for determining whether MPCpr can be used as a guide for
choosing and eventually administering fluoroquinolones to significantly reduce the development of resistance.
| |
INTRODUCTION |
Antibiotic resistance among human
pathogens now occurs in almost every bacterial species for which
antibiotic therapies exist (15, 16, 26). In the case of
Streptococcus pneumoniae, resistance to penicillin and
erythromycin has become so widespread that clinicians have started to
use the fluoroquinolones for therapy. As a result, drug resistance is
now emerging against the quinolones (4). More active
fluoroquinolones are becoming available, but new treatment strategies
must accompany use of those agents to halt the selection of resistant
mutants before the entire quinolone class of drugs becomes ineffective.
It has been suggested that if bacterial cells must attain two
concurrent resistance mutations for growth in the presence of a
quinolone, then few mutants would be selectively amplified because double mutations should rarely occur (19, 21, 28, 29). For
example, bacterial populations may reach 1010 cells in
human infections (1, 9, 15), but at a mutation frequency
of 10
7, more than 1014 bacteria
(107 × 107) would be required to detect
two concurrent fluoroquinolone-resistant mutations. When we examined
the effect of fluoroquinolone concentration on the selection of
resistant mutants of Mycobacterium bovis BCG and
Staphylococcus aureus, we found a concentration with each organism at which no mutant was recovered when more than
1010 cells were applied to agar plates (5).
This drug concentration, which we designated as the mutant prevention
concentration (MPC), would require a bacterial cell to develop more
than one resistance mutation for growth. Thus at concentrations above
the MPC, a bacterial population size greater than that normally present
during infection would be necessary to observe outgrowth of a resistant
mutant. Since fluoroquinolone structure affects the value of the MPC
(5), it appeared that the MPC might serve as a simple
measure of antibiotic potency that incorporates the ability of a
compound to restrict selection of resistant mutants (28).
In principle MPC also represents a dosing threshold above which mutants
should rarely arise; use of MPC would add consideration of the
development of resistance to the traditional goal of clearing infection. With a clinical isolate of Mycobacterium
tuberculosis, we found that MPCs for two new C-8-methoxy
fluoroquinolones are below the maximum attainable drug concentration in
serum (6). Thus the possibility exists that
fluoroquinolones might be administered such that serum drug
concentrations in patients exceed the MPC. Whether this is true for
other quinolone-pathogen combinations and for large numbers of clinical
isolates is unknown.
Examination of large numbers of clinical isolates generally involves
measurement of antibiotic potency in terms of the MIC. With the agar
dilution method, approximately 105 CFU is applied to each
of a series of agar plates containing various antibiotic concentrations
(18). The concentration that allows no colony formation is
taken as the MIC. Measurement of MPC is carried out using the same
strategy except that more cells, on the order of 1010, are
applied to agar plates. Consequently, it should be possible to perform
MPC measurements for a large number of clinical isolates. Those
measurements could then be compared with published values of
pharmacokinetic parameters to determine whether and for how long the
serum drug concentration would be above the MPC. The relationship
between pharmacokinetics and MPC could then be used to identify
compounds for further examination of the inability to restrict
selection of resistance.
In the present work, we tested five fluoroquinolones (gatifloxacin,
grepafloxacin, levofloxacin, moxifloxacin, and trovafloxacin) with
clinical isolates of S. pneumoniae. A hierachy of potency was clear. For the most active compounds, moxifloxacin and
gatifloxacin, a provisional MPC (MPCpr), which
overestimates MPC by about twofold, was below the maximum serum drug
concentrations attained with the doses recommended for streptococcal
pneumonia. Thus these fluoroquinolones may be useful in clinical trials
to determine the utility of MPC in reducing the development of
resistance. To facilitate further testing of MPC, we describe an
empirical relationship that will allow MPC to be calculated from
standard MIC measurements made by the agar dilution method.
| |
MATERIALS AND METHODS |
Bacterial strains and bacteriological methods.
Isolates of
S. pneumoniae were obtained from the Clinical Microbiology
Division, Royal University Hospital, Saskatoon, Saskatchewan, Canada.
No preselection criterion was used that would favor inclusion or
exclusion of resistant isolates, and care was taken to avoid obtaining
more than one specimen from a given patient. The MIC was determined by
the standard twofold agar dilution method (18).
For MPC measurements, starter cultures were spread on blood agar plates
(six plates per isolate) (PML, Richmond, Canada) and incubated
overnight (18 to 24 h) at 35 to 37°C in 5% CO2.
Bacterial cells were then transferred from the plates to 500 ml of
Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.), followed by
overnight incubation at 35 to 37°C in 5% CO2. After
incubation, cultures were estimated to have concentrations of 
3 × 108 CFU per ml by turbidity measurements. Cultures were
concentrated by centrifugation at 5,000 × g for 30 min
and resuspended in 3 ml of Todd-Hewitt broth. Aliquots of 200 µl,
containing 1010 CFU, were applied to tryptic soy agar
plates containing 5% sheep red blood cells (18). For each
experiment, agar dilution plates were prepared by incorporating
fluoroquinolones at seven concentrations into the tryptic soy
agar-sheep red blood cell plates (plates were stored at 4°C and used
within 7 days of preparation). Each experiment included the fully
susceptible control strain S. pneumoniae ATCC 49619. Inoculated plates were incubated for 24 h at 35 to 37°C in 5%
CO2 and then screened for growth. All plates were
reincubated for an additional 24 h and reexamined.
MPCpr was recorded as the lowest antibiotic concentration
that allowed no growth. In a few cases a thin film was observed after
incubation on plates with high fluoroquinolone concentrations. When
these plates were washed with a growth medium that was reapplied to
drug-free agar, no growth was observed. These plates were scored as
negative for growth. All MPCpr determinations were made in
duplicate, and the results were identical.
Fluoroquinolones.
Sources of the fluoroquinolones were Bayer
AG (moxifloxacin), Bristol-Myers Squibb (gatifloxacin), Glaxo-Wellcome
(grepafloxacin), Johnson-Ortho (levofloxacin), and Pfizer, Inc.
(trovafloxacin). Powdered forms of each compound were dissolved
according to manufacturers' instructions. Stock solutions were used as
fresh preparations or from samples stored at 
70°C.
DNA isolation, amplification and nucleotide sequence
determination.
Selected isolates of S. pneumoniae were
grown on brain heart infusion agar (Difco) containing 10% defibrinated
sheep blood (Hemostat Laboratories, Dixon, Calif.) following
high-density inoculation. Incubation was overnight at 37°C with 5%
CO2. Bacteria grown as confluent lawns were recovered from
agar plates by washing with 2 ml of Todd Hewitt broth per plate. Cells
were concentrated by centrifugation, washed once with lysis buffer (50 mM Tris-HCl [pH 8.0] and 5 mM EDTA), and resuspended in 400 µl of
lysis buffer per plate. Then 50 µl of 10% sodium dodecyl sulfate and
20 µl of proteinase K (10 mg/ml) were added, and the mixture was
incubated, first at 55°C for 30 min and then at 37°C for 1.5 h. Cell lysates were extracted with phenol, then with phenol:chloroform
(1:1), and finally with chloroform. DNA was precipitated with ethanol and recovered by centrifugation. DNA was then dissolved in Tris-EDTA buffer (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA) and treated with a
final concentration of 100 µg of RNase A per ml for 1 h at
37°C. DNA was reprecipitated with ethanol and dissolved in Tris-EDTA buffer. The nucleotide sequences of the
quinolone-resistance-determining regions of parC and
gyrA were determined with an automated DNA sequencer using
primer SP-parC.seq (5' TCA GCG CCG TAT TCT TTA TTC TAT
G 3') and primer SP-gyrA.seq (5' TCG AGA TGG CTT
AAA ACC TGT TCA C 3') after PCR amplification of DNA fragments
using primers SP-parCfwd (5' GTC TAA CAT
TCA AAA CAT GTC CCT G 3'), SP-parCrev (5' TCT TTC TCC GTA TCG TCA AAG TTC 3') for parC
and SP-gyrAfwd (5' TGT CAA TCT GAC AAA GGA
GAT GAA G 3'), and SP-gyrArev (5' CCA
GTT GCT CCA TTA ACC AAA AG 3') for gyrA.
| |
RESULTS AND DISCUSSION |
Estimation of MPC.
In preliminary work with S. pneumoniae strain ATCC 49619, we found that moxifloxacin-resistant
mutants are recovered only within a narrow, twofold drug concentration
range even when more than 1010 cells were examined. A
similar finding was obtained previously with another strain
(23) and with clinafloxacin, a C-8-chlorine fluoroquinolone (20). A narrow drug concentration range
for mutant selection led us to expect that agar plates used in a
standard twofold dilution analysis with clinical isolates would exhibit either confluent growth or no colonies, at least for the C-8-methoxy compounds moxifloxacin and gatifloxacin. When 1010 cells
were applied per plate, a sharp drop in growth was seen over a range of
one dilution. When the minimal concentration at which no colony was
recovered (MPCpr) was plotted against the number of
isolates, distinct peaks were seen in the distribution (Fig.
1). The peak for moxifloxacin appeared at
the lowest drug concentration; therefore, moxifloxacin was the most
potent fluoroquinolone by this assay.
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FIG. 1.
Distribution of S. pneumoniae isolates
with respect to MPCpr. MPCpr was
determined for each isolate by the agar dilution method. White areas of
bars represent isolates containing parC mutations known to
confer resistance; shaded regions represent isolates containing
parC mutations that have not been demonstrated by genetic
tests to confer quinolone resistance; solid regions represent
unsequenced isolates. Dashed line is for alignment of panels. Panels:
A, moxifloxacin; B, gatifloxacin; C, trovafloxacin; D, grepafloxacin;
and E, levofloxacin.
|
|
We consider the values of MPC
pr shown in Fig.
1 to be
provisional because the high inocula used could have raised the drug
concentration required to prevent the isolation of mutants (e.g.,
crowding may have interfered with access of the fluoroquinolone
to the
cells). To test this idea, four isolates were examined
by applying
10
8 cells to each of 100 agar plates at a concentration of
moxifloxacin
or levofloxacin that was one dilution lower than that of
MPC
pr.
In each case no growth was observed, indicating that
MPC
pr overestimates
MPC. These data also argue against
complications due to autolysis
occurring at high cell density, since
that would have made MPC
pr an underestimation of MPC. In a
separate study using a laboratory
isolate of
S. pneumoniae
(ATCC 49619), various fluoroquinolone
concentrations were tested with
dilute bacterial cultures and
many agar plates so that mutants were
recovered as single colonies.
The MPC was 0.5 µg of moxifloxacin per
ml and 2.6 µg of levofloxacin
per ml, respectively (X. Li, X. Zhao,
and K. Drlica, unpublished
observation). With this
S. pneumoniae strain, the MPC
pr was 1
µg of
moxifloxacin per ml and 4 µg of levofloxacin per ml, using
the
twofold agar dilution assay described in Materials and Methods
(data
not shown). Consequently, MPC
pr overestimates MPC by about
twofold for both the most active and the least active compounds
in the
present study. This small inoculum effect was deemed acceptable
for the
present analysis because the ability to load more cells
on agar plates
greatly reduced the number of plates necessary
for examination of large
numbers of
isolates.
Since working with large numbers of
S. pneumoniae is
cumbersome, we determined an empirical relationship between
MPC
pr and
the MIC of the drug so that the MPC
pr
of a drug can be calculated
from the MIC of the drug. When the MIC of a
drug was measured
by the agar dilution method, most of the isolates
exhibited a
four- to eightfold difference between the MIC and
MPC
pr (two to
three dilutions) of the five fluoroquinolones
tested (Table
1).
The ratio was slightly
higher for trovafloxacin and grepafloxacin.
When the ratio of the
MPC
pr to the MIC of a drug was calculated
using values that
exceeded those for 90% of the isolates (MPC
pr90 and
MIC
90), the ratio was 8 for three of the compounds and 16
for trovafloxacin and grepafloxacin (Table
2). Similar ratios
were obtained for the
MPC
pr and the MIC of these drugs with the
laboratory strain
ATCC 49619 (data not shown). When cells were
distributed to more agar
plates and when smaller quinolone concentration
increments were used to
obtain single resistant colonies. The
MPC/MIC ratio for moxifloxacin
and levofloxacin with strain ATCC
49619 was lower (three- to fourfold,
using an MIC of the drugs
that blocks growth of 99% of the CFUs; X. Li, X. Zhao, and K.
Drlica, unpublished observation). We conclude that
the ratios
listed in Table
2 are overestimates of the ratio of the MPC
to
the MIC of the drugs. Therefore, using the numbers in Table
2 to
calculate the MPC
pr from the MIC of the drugs will provide
a conservative estimate of MPC. Additional studies are required
with
other
S. pneumoniae populations to determine whether the
ratios we calculated are generally applicable to this bacterial
species.
Some of the increased incidence of fluoroquinolone-resistant
S. pneumoniae (
4) has been associated with treatment of
penicillin-resistant
cases of pneumonia (discussed in
4).
For the newer fluoroquinolones
the value of MIC
90 is
unaffected by pneumococcal resistance to
penicillin and/or other agents
(
3). Likewise, no increase in
MPC
pr was
associated with the presence of penicillin resistance
in the present
data set (data not shown). Thus potential cross-resistance
between the
two classes of compounds does not appear to be a
problem.
Fluoroquinolone-resistant mutants.
Some of the isolates
examined at high inoculum concentrations required exceptionally high
concentrations of fluoroquinolone to prevent colony formation (Fig. 1,
open and shaded areas of bars). Since ciprofloxacin and levofloxacin
have been used extensively in Canada as therapy for streptococcal
pneumonia and since many fluoroquinolone-resistant isolates have
emerged (4), we suspected that at least some of the
isolates in the present study contained mutations in the target genes,
parC (topoisomerase IV) and/or gyrA (gyrase). To
test this idea, DNA was obtained from 22 isolates, and the nucleotide
sequences of the quinolone-resistance-determining regions of the two
genes were determined. As shown in Table
3, 17 isolates were parC
mutants, 7 of which also contained a gyrA mutation. Six of
the parC mutants (isolate numbers 10, 12, 13, 15, 16, and
18) contained alleles known from genetic studies (10, 11, 16,
21) to confer resistance (predicted amino acid changes of Ser-79
to Phe and Asp-83 to Asn). Of these, three also contained a GyrA
alteration (Ser-81 changed to Phe or Tyr) known to confer resistance.
Since MPC is based on the recovery of colonies grown from wild-type
populations (5), the six resistant mutants (white portions
of bars in Fig. 1) were excluded from determination of MPCpr90 (Table 2). For 11 other isolates the nucleotide
sequence predicted changes of Ser-52 to Gly, Asn-91 to Asp, or Lys-137 to Asn in the ParC protein (shaded portion of bars in Fig. 1). To our
knowledge, genetic studies have not been performed that attribute
resistance to these alleles. Consequently, we did not exclude these
strains or ones lacking a parC or gyrA mutation from the determination of MPCpr90. These isolates may
contribute to the finding that MPCpr90 in Table 2
overestimates MPC.
When the average MIC for the resistant isolates was determined, the
five fluoroquinolones could be ranked in terms of potency,
in
descending order, with moxifloxacin = trovafloxacin > gatifloxacin
> grepafloxacin > levofloxacin (not shown). A
similar order in
potency was seen with a separate collection of
resistant mutants
(
12), although in that case
grepafloxacin was, on average, more
potent than gatifloxacin. Variants
with identical predicted amino
acid changes occasionally exhibited
different response patterns
of susceptibility to the five
fluoroquinolones (Table
3). These
differences, which were reproducible,
probably arose from unidentified
mutations present in some isolates but
absent from
others.
Relationship of MPCpr to pharmacokinetics.
For MPC
to be a therapeutically useful parameter, its value must be below the
serum and tissue drug concentrations attained following administration
of drug doses that are safe for patients. Recommended doses and
pharmacokinetic parameters for the five fluoroquinolones are listed in
Table 4. Since trovafloxacin and grepafloxacin have been withdrawn from the market, they are not considered further here. Moxifloxacin, the most potent of the compounds
tested, has a maximum serum drug concentration of 4.5 µg/ml, about
twice that of MPCpr for more than 90% of the isolates. Since the half-life of moxifloxacin is 12 h, daily dosing should keep concentrations of moxifloxacin in serum above the
MPCpr for most of the treatment time. This should help
restrict the selection of resistant mutants. Since MPCpr
overestimates MPC by about twofold and since quinolone concentration in
relevant tissue may be higher than in serum (J. Andrews, D. Honeybourne, G. Jevons, and R. Wise, Abstr. 38th Intersci. Conf.
Antimicrob. Agents Chemother., abstr. A29, 1998), gatifloxacin may also
restrict the selection of resistant mutants, especially if administered
twice daily. We speculate that levofloxacin may require higher doses,
perhaps administered twice daily, to attain the same potency with
respect to restricting selection of resistant mutants. Clinical trials
are now required to test these ideas.
The serum drug concentrations listed in Table
4 represent total
concentrations. Fluoroquinolones bind to serum proteins,
so additional
corrections may be necessary when particular compounds
are compared.
The extent of binding varies among the compounds,
but in general less
than half of the total drug that is present
is bound (
27);
consequently, protein binding probably has little
effect on the
conclusions reached above. A future refinement may
utilize bioassays
rather than high-pressure liquid chromatography
to determine relevant
concentrations.
Conclusions.
The endpoint of antibiotic dosing strategies,
such as those based on area under the inhibitory concentration curve
(reviewed in 24), has often been clearance of infection,
measured either as survival in animal models or loss of symptoms in
humans. Widespread development of resistance suggests that
consideration should also be given to restricting the selection of
resistant mutants. One approach has utilized the ratio of maximum
concentration of drug in serum to the MIC of the drugs; values have
been found that restrict mutant outgrowth in vitro (2) and
correlate with animal survival (7). However, this ratio
does not take into account the time at which concentration is high,
which may be important for preventing the development of resistance.
Consequently, we have been examining the possibility of using a new
potency parameter termed the MPC, the concentration that allows no
growth of first-step mutants. Compounds and dosing protocols can
readily be compared for the time that drug concentration in tissue
exceeds the MPC.
The data described above show that with
S. pneumoniae
MPC
pr can be below the serum drug concentration that can be
safely achieved
for moxifloxacin and gatifloxacin. This may also be
true for levofloxacin
if the compound is administered more frequently
and at higher
levels. Thus one of the criteria has been met for using
the MPC
to slow the development of quinolone resistance. Whether
keeping
relevant tissue concentrations of this drug above the MPC will
actually restrict the selection of mutants in animal models or
in human
patients has not been
determined.
For some antibiotic-pathogen combinations, the MPC may not fall below
tissue drug concentrations that are achievable with
safe doses.
Although the antibiotic often may clear infection,
for such cases
enrichment of resistant mutants will gradually
erode the utility of the
agent, especially if administered to
tens of millions of patients each
year. Such antibiotics can be
preserved by combination therapy if such
therapy is instituted
before resistance becomes too extensive (for
discussion see
28).
Measurement of MPC with
S. pneumoniae is cumbersome, so we
determined an empirical relationship with the MIC of the
fluoroquinolones
examined, a more readily determined parameter. Use of
this relationship
should help determine whether use of a given compound
as therapy
against a particular population of
S. pneumoniae
will restrict
the development of resistance, provided that no silent
target
mutations are present (see below). It may be possible to extend
this conclusion to individual isolates when susceptibility measurements
from clinical laboratories can be related to the MIC of different
drugs
as determined by agar
dilution.
An important question is whether moxifloxacin- or gatifloxacin-based
therapy for
S. pneumoniae infection is influenced by
the
continued use of older compounds such as ciprofloxacin and
levofloxacin. The latter agents have topoisomerase IV as their
primary
target, while the primary target for gatifloxacin and
moxifloxacin is
gyrase (
10,
22,
23). This difference in
target means that
first-step mutants selected by ciprofloxacin
and levofloxacin may
remain susceptible to gatifloxacin and moxifloxacin
(
22).
However, we have shown with
Escherichia coli that a
parC resistance allele, which has no effect on the MIC of
the drugs,
can increase by orders of magnitude the frequency at which
resistant
mutants are selected by C-8-methoxy fluoroquinolones
(
29). The
effect of the
parC mutation is to
require only one resistance
mutation rather than two for bacterial
growth in the presence
of high concentrations of the C-8-methoxy
compound. This principle
appears to hold for
S. pneumoniae
(X. Li, X. Zhao, and K. Drlica,
unpublished observation and
23). Thus we expect continued use
of ciprofloxacin and
levofloxacin to seriously shorten the useful
lifespan of moxifloxacin
and gatifloxacin; although the latter
two compounds may be potent
enough to treat infection caused by
parC mutants (selected
by ciprofloxacin and levofloxacin),
parC gyrA double mutants
would be readily selected by moxifloxacin
or gatifloxacin if the
pathogens already contain a
parC resistance
allele. Thus,
slowing the development of resistance may involve
careful management of
compounds within the same general
class.
| |
ACKNOWLEDGMENTS |
We thank J. de Azavedo, M. Gennaro, and S. Kayman for critical
comments on the manuscript, D. Leciuk for excellent clerical assistance, and S. Borsos for technical support.
This work was supported by grants to J.B. from Bayer AG and to K.D.
from the National Institutes of Health (AI35257) and Bayer AG.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Clinical Microbiology, Royal University Hospital, 103 Hospital Ave., Saskatoon, Saskatchewan S7N 0W8, Canada. Phone: (306) 655-6943. Fax:
(306) 655-6947. E-mail: blondeauj{at}sdh.sk.ca.
| |
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Antimicrobial Agents and Chemotherapy, February 2001, p. 433-438, Vol. 45, No. 2
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.2.433-438.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
