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Antimicrobial Agents and Chemotherapy, May 1999, p. 1177-1182, Vol. 43, No. 5
Department of Pathology (Clinical
Microbiology), Hershey Medical Center, Hershey, Pennsylvania
17033,1 and Department of Pathology
(Clinical Microbiology), Case Western Reserve University,
Cleveland, Ohio 441062
Received 17 September 1998/Returned for modification 4 December
1998/Accepted 11 February 1999
The ability of 50 sequential subcultures in subinhibitory
concentrations of ciprofloxacin, levofloxacin, grepafloxacin,
sparfloxacin, trovafloxacin, and amoxicillin-clavulanate to select for
resistance was studied for six penicillin-susceptible and four
penicillin-intermediate pneumococci. Subculturing in ciprofloxacin,
grepafloxacin, levofloxacin, and sparfloxacin led to selection of
mutants requiring increased MICs for all 10 strains, with MICs rising
from (i) 0.5 to 4.0 to (ii) 4.0 to 32.0 µg/ml after 7 to 12 passages
for ciprofloxacin, from (i) 0.06 to 0.25 to (ii) 0.5 to 8.0 µg/ml
after 5 to 23 passages for grepafloxacin, from (i) 0.5 to 1.0 to (ii)
4.0 to 64 µg/ml after 14 to 49 passages for levofloxacin, and from
(i) 0.125 to 0.25 to (ii) 1.0 to 16.0 µg/ml after 8 to 26 passages
for sparfloxacin. Subculturing in trovafloxacin led to increased MICs
for eight strains, with MICs rising from (i) 0.06 to 0.125 to (ii) 0.5 to 8.0 µg/ml after 6 to 28 passages. Subculturing in
amoxicillin-clavulanate led to raised MICs for only one strain, with
the MIC rising from 0.015 to 0.125 µg/ml after 24 passages. Double
mutations in both ParC and GyrA led to high-level quinolone resistance
when ParC mutations were at S79. Trovafloxacin MICs were 1 to 2 µg/ml
in double mutants with ParC mutations at positions other than S79 (e.g., D83). Mutations in ParE (at D435, R447, and E474) and GyrB (at
S405, D406, and D435) were found in four and six mutants, respectively.
In the presence of reserpine, 29 mutants had lower ciprofloxacin MICs
(2 to 16 times lower), 8 mutants had lower levofloxacin MICs (2 times),
and one mutant had a lower trovafloxacin MIC (2 times), suggesting the
involvement of an efflux mechanism. In contrast to the case for
quinolones, subculturing in the presence of amoxicillin-clavulanate did
not select for resistance to this drug.
Amoxicillin-clavulanate, a
combination of a penicillin and a Presently, the incidence of quinolone resistance in pneumococci is low.
In a study by Felmingham in 1998 (6) examining 4,665 pneumococcal strains from 1992 to 1996, the incidences of resistance to
ciprofloxacin (MIC > 4 µg/ml) and ofloxacin (MIC > 4 µg/ml) were 0.6 and 0.5%, respectively. In another study in France
examining 4,804 strains from 1996, the incidence of resistance to
ofloxacin was 0.79%. In a study presented by Jacobs et al. examining
over 1,400 pneumococcal strains from the United States in 1997, there
were none that were resistant to ciprofloxacin (9).
The primary targets of fluoroquinolones are topoisomerase IV and DNA
gyrase. Topoisomerase IV is composed of two C (ParC) and two E (ParE)
subunits, which are encoded by parC and parE, respectively, and DNA gyrase is composed of two A (GyrA) and two B
(GyrB) subunits, which are encoded by gyrA and
gyrB, respectively. Resistance to fluoroquinolones in
pneumococci usually occurs in a stepwise fashion, with low-level
resistance caused by mutations in the quinolone resistance-determining
region (QRDR) of parC and high-level resistance occurring
after an additional mutation in the QRDR of gyrA (10,
15, 16, 21), except for sparfloxacin, which has been reported to
target primarily GyrA (17). Mutations in the QRDRs of
parE and gyrB are also believed to play a role in
fluoroquinolone resistance (10, 16, 19).
The recent dramatic increase in the incidence of drug-resistant
pneumococci may be due in part to abuse of oral drugs such as
cephalosporins and macrolides (18). In a previous
study (18), we found that sequential subcultures in
subinhibitory concentrations of amoxicillin-clavulanate did not lead to
increased pneumococcal MICs, in contrast to the case for azithromycin
and, to a lesser extent, cefuroxime and cefaclor.
Fluoroquinolone resistance can be selected for in vitro in pneumococci
(8, 10, 16, 21), and this must be considered during therapy.
In order to shed further light on the capability of drugs potentially
used for treatment of the same infections to select for raised MICs for
pneumococci, we repeatedly exposed 10 strains of Streptococcus
pneumoniae to subinhibitory concentrations of ciprofloxacin,
grepafloxacin, levofloxacin, sparfloxacin, trovafloxacin, and
amoxicillin-clavulanate to determine if resistance developed. The
mutations in parC, parE, gyrA,
and gyrB associated with quinolone resistance were also
determined. In addition, many of the mutants were checked for the
presence of a quinolone efflux mechanism by comparing MICs in the
presence and absence of reserpine (a known efflux pump inhibitor)
(4).
Bacteria and antimicrobial agents.
Ten strains of S. pneumoniae isolated within the past 5 years were randomly
selected. Organisms were identified by optochin susceptibility and
classified by serotyping. Six were susceptible to penicillin
(MICs of MIC determination.
MICs were determined by standardized
microdilution methodology in Mueller-Hinton broth (Difco Laboratories)
supplemented with 5% lysed horse blood (13). Breakpoints
for all compounds except ciprofloxacin were those approved by the
National Committee for Clinical Laboratory Standards (14),
and susceptibility breakpoints are as follows: grepafloxacin, Serial passages.
Glass tubes containing 1 ml of
cation-adjusted Mueller-Hinton broth (Difco) supplemented with 5%
lysed horse blood with doubling antibiotic dilutions were inoculated
with approximately 5 × 105 CFU/ml at antibiotic
concentrations from 3 doubling dilutions above to 3 doubling dilutions
below the MIC of each agent for each strain. The initial inoculum was
prepared by suspending growth from an overnight Trypticase soy blood
agar plate (Difco) in Mueller-Hinton broth. Tubes were incubated at
35°C for 24 h. Daily passages were then performed for 50 days by
taking an inoculum from the tube nearest the MIC (usually one tube
below) which had the same turbidity as the antibiotic-free controls.
Periodically for some of the mutants, an aliquot from a tube used as an
inoculum was frozen in double-strength skim milk at Serotyping.
Serotyping of parent and passaged strains was
performed by the standard Quellung method with sera from Statens
Seruminstitut (Copenhagen, Denmark).
PFGE.
To determine whether resistant isolates obtained at
the end of serial passages were derived from those tested at the
beginning of the study, the parent strains and the strains with
increased MICs obtained after the last passage were tested by
pulsed-field gel electrophoresis (PFGE) with a CHEF DR III apparatus
(Bio-Rad, Hercules, Calif.). Bacterial cultures were grown for 6 h
at 37°C (in 5% CO2) in 5 ml of Todd-Hewitt broth
supplemented with 5% yeast extract (BBL Microbiology Systems,
Cockeysville, Md.). Cell pellets were collected by centrifugation of
approximately 1.5 ml of culture for 30 s at 21,000 × g. Cell pellets were then resuspended in 150 µl of Pett IV
buffer (10 mM Tris-HCl [pH 7.6], 1 M NaCl) and warmed to 55°C. An
equal volume of 2% Incert agarose (FMC, Rockland, Maine) in distilled
H2O was added to the warmed cells, and 100 µl of the
cell-agarose mixture was distributed into a plug mold and allowed to
solidify. Plugs were incubated for 1 h at 37°C in 1 ml of lysis
buffer (6 mM Tris-HCl [pH 7.4], 1 M NaCl, 10 mM EDTA [pH 8.0],
0.2% deoxycholate, 0.5% sodium lauroyl sarcosine) to which lysozyme
(Sigma, St. Louis, Mo.) at 0.5 mg/ml and lysostaphin (Sigma) at 0.05 mg/ml were added fresh. The lysis solution was replaced with 300 µl
of ESP (10 mM Tris-HCl [pH 7.4], 1 mM EDTA, 1% sodium dodecyl
sulfate), to which proteinase K (Sigma) at a final concentration of 8 U/ml was added before use. The plugs were incubated overnight at
55°C. ESP was decanted, and the plugs were washed three times with 1 ml of TE (10 mM Tris-HCl [pH 7.4], 0.1 mM EDTA) and then stored in 1 ml of TE at 4°C. Agarose-embedded DNA was digested to completion with
20 U of SmaI (New England Biolabs, Beverly, Mass.) for
6 h at room temperature, and DNA fragments were separated as
described by Moissenet et al. (11).
PCR of quinolone resistance determinants and DNA sequence
analysis.
To determine whether strains that developed resistance
to quinolones had alterations in topoisomerase IV or DNA gyrase
compared to the parent strains, parC, parE,
gyrA, and gyrB were amplified by the PCR method
with the primers and cycling conditions described by Pan et al.
(16). Template DNA for PCR was prepared as follows. A colony
from overnight growth was lysed by incubation for 1 h at 37°C in
lysis buffer (see "PFGE" above for details), and DNA was isolated
by using a Prep-A-Gene kit (Bio-Rad) as recommended by the
manufacturer. After amplification, PCR products were purified from
excess primers and nucleotides by using a QIAquick PCR purification kit
as recommended by the manufacturer (Qiagen, Valencia, Calif.) and
sequenced directly by using an Applied Biosystems model 373A DNA
sequencer. Mutants with mutations widely described in the literature (e.g., Ser79 Determination of efflux mechanism.
MICs were determined in
the presence and absence of 10 µg of reserpine (Sigma) per ml as
described previously (4) with 47 mutant strains for which
the MIC of a particular quinolone (after 10 subcultures on drug-free
medium) was at least fourfold greater than the corresponding MIC for
the parent strain. An efflux mechanism was believed to be present when
the MIC in the presence of reserpine was at least twofold less (1 doubling dilution) than the MIC in the absence of reserpine (tests
were repeated three times).
Subculturing in subinhibitory concentrations of antibiotic.
MIC results from subculturing in subinhibitory concentrations of
antibiotics are summarized in Table 1.
Subculturing in amoxicillin-clavulanate led to increased
MICs for only one strain, with the MIC rising from 0.015 to 0.125 µg/ml after 24 subcultures. MICs of ciprofloxacin, grepafloxacin,
levofloxacin, sparfloxacin, and trovafloxacin were unaffected for this
strain.
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
In Vitro Development of Resistance to Five
Quinolones and Amoxicillin-Clavulanate in Streptococcus
pneumoniae
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamase inhibitor, is the
most active oral -lactam agent overall against pneumococci
(including those for which the penicillin MICs are increased),
-lactamase-positive and -negative Haemophilus influenzae, and Moraxella catarrhalis. In view of
the increased incidence of community-acquired respiratory tract
infections caused by penicillin-resistant pneumococci (1,
5), amoxicillin-clavulanate may be used as empiric therapy
of sinusitis, acute exacerbations of chronic bronchitis, and
other respiratory tract infections caused by these organisms,
where chlamydiae, mycoplasmas, and legionellae are not
involved. Levofloxacin, grepafloxacin, sparfloxacin, and
trovafloxacin are quinolones with broader spectra than
ciprofloxacin (2, 7, 20, 22), especially against
pneumococci; this allows them to be used as alternate empiric therapy
to amoxicillin-clavulanate for the same infections mentioned above.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
0.06 µg/ml), and four showed intermediate penicillin
resistance (MICs of 0.125 to 0.25 µg/ml). Strains were stored at 
70°C in double-strength skim milk (Difco Laboratories, Detroit, Mich.) before being tested. Antimicrobials were obtained as follows: amoxicillin-clavulanate from SmithKline Beecham
Laboratories, Collegeville, Pa.; ciprofloxacin from Bayer, Inc., West
Haven, Conn.; levofloxacin from RW Johnson Pharmaceutical Research
Institute, Raritan, N.J.; sparfloxacin from Rhône-Poulenc Rorer,
Collegeville, Pa.; grepafloxacin from Glaxo-Wellcome, Inc., Research
Triangle Park, N.C.; and trovafloxacin from Pfizer, Inc., New York,
N.Y.
0.5
µg/ml; sparfloxacin, 0.5 µg/ml; trovafloxacin, 1 µg/ml; and
levofloxacin, 2 µg/ml. Strains were considered susceptible to
ciprofloxacin when MICs were 2 µg/ml.
70°C for later
analysis (MIC testing and sequencing of parC and
gyrA genes). When the MIC for a strain increased fourfold,
irrespective of the number of subcultures, passaging was stopped and
strains were subcultured in antibiotic-free medium for 10 serial
passages. A maximum of 50 serial passages in antibiotic were performed.
Tyr or Phe in ParC and Ser83Tyr or Phe in GyrA) were sequenced once in the forward direction. Mutants with no
mutations in a particular gene or with a previously undescribed mutation were sequenced twice in the forward direction and once in the
reverse direction on products of independent PCRs.
RESULTS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Resistance selection results
Cross-resistance among mutants.
Of 48 mutants for which MICs
of at least one of the quinolones were elevated, 33 mutants were
resistant in vitro to ciprofloxacin, 31 were resistant to
grepafloxacin, 34 were resistant to sparfloxacin, 28 were
resistant to levofloxacin, and 9 were resistant to trovafloxacin (Table
2).
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1 µg/ml) were also cross resistant to sparfloxacin and in
most cases to ciprofloxacin, while cross-resistance to levofloxacin and
trovafloxacin did not occur until the selected strain's grepafloxacin MIC reached 4 and 8 µg/ml, respectively. Mutant strains that were selected in levofloxacin (MICs of 4 µg/ml) were cross
resistant to ciprofloxacin, grepafloxacin, and sparfloxacin in
most cases (strain 5 remained susceptible to ciprofloxacin, and
strain 10 remained susceptible to grepafloxacin and sparfloxacin),
while cross-resistance to trovafloxacin did not occur until the
selected strain's levofloxacin MIC reached 16 µg/ml. Mutant
strains that were selected in sparfloxacin (MICs of 1 µg/ml)
were in most cases cross resistant to grepafloxacin, while resistance
to ciprofloxacin, levofloxacin, and trovafloxacin did not occur until
the selected strain's sparfloxacin MIC reached 2, 2, and 4
µg/ml, respectively. Mutant strains that were selected in
trovafloxacin (MICs of 2 µg/ml) were cross resistant to all the
other quinolones tested. For all strains, subculturing in any of
the quinolones tested did not affect amoxicillin-clavulanate MICs.
Serotyping and PFGE. The 10 pneumococcal strains used comprised serotypes 1, 6A, 6B, 14, and 19F. All mutants had serotypes identical to those of the parent strains, and all but two had PFGE patterns identical to those of the parent strains. Strain 4 and strain 7, which were selected in ciprofloxacin and levofloxacin, respectively, had a one-band difference in PFGE patterns compared to the parent strains, which was most likely caused by a point mutation in one of the SmaI restriction sites.
Mutations in topoisomerase IV and DNA gyrase.
Mutations in the
QRDR that led to an amino acid change are listed in Table 1. Most
mutations were present in ParC and/or GyrA, while few mutations were
present in ParE and GyrB. Parent strains 4, 6, and 8 had a Lys137Asn
substitution in ParC compared to wild-type sequences (15,
16); however, this variation has been shown not to affect
ciprofloxacin MICs (12). Additionally, parent strains 1, 3, 7, 8, and 10 had an Ile460Val substitution in ParE. Parent
strain 8 had both mutations in ParC and ParE and also had the highest
ciprofloxacin MIC (4 µg/ml) of the 10 parent strains. In all
other cases the amino acid sequences for ParC, ParE, GyrA, and GyrB
from the parent strains were identical to the wild-type sequences
(15, 16).
Tyr or Phe in ParC and either
Ser83Phe or Glu87Lys or Gln in GyrA were associated with
high-level resistance to all of the fluoroquinolones tested. However,
when the ParC mutation was at a position other than Ser79 (e.g., D83),
trovafloxacin MICs were 1 to 2 µg/ml while the MICs of the other
fluoroquinolones remained high. Strain 4 exposed to trovafloxacin had
mutations of Ala115Val in ParC and Ser83Tyr in GyrA yet remained
susceptible to all the fluoroquinolones except sparfloxacin, to which
it had low-level resistance (MIC, 1 µg/ml).
Mutants with a single mutation only in ParC (Ser79Phe or Tyr) showed
various susceptibility patterns, including (i) susceptibility to all
quinolones (e.g., strain 7 exposed to grepafloxacin or trovafloxacin),
(ii) resistance only to ciprofloxacin (e.g., strain 6 exposed to
ciprofloxacin), (iii) resistance to all quinolones except trovafloxacin
(e.g., strain 8 exposed to ciprofloxacin or grepafloxacin), and (iv)
resistance to all quinolones (e.g., strain 2 exposed to sparfloxacin).
Mutants with a single mutation only in GyrA (Ser83Phe or Tyr) were
associated with resistance to grepafloxacin and sparfloxacin in all
cases and also with resistance to ciprofloxacin and levofloxacin in
many cases. There were not any mutants with a mutation only in GyrA
that were resistant to trovafloxacin.
Mutations in ParE and GyrB were observed in four and six mutants,
respectively. Three of the four mutants with ParE mutations and four of
the six mutants with GyrB mutations were derived from strains exposed
to levofloxacin. Two mutants had a mutation only in GyrB (strain 5 exposed to sparfloxacin and strain 6 exposed to grepafloxacin). These
two mutants had increases primarily in grepafloxacin and sparfloxacin
MICs. Of five mutants with mutations in both (i) ParE or GyrB and (ii)
ParC or GyrA (e.g., strains 4 and 6 exposed to levofloxacin), four were
resistant to all of the quinolones except trovafloxacin and one was
resistant to all of the quinolones. One mutant (strain 2 exposed to
levofloxacin) had mutations only in ParE and GyrB (at position Asp435)
and was resistant to all quinolones except trovafloxacin.
Most mutants with no mutations in ParC, ParE, GyrA, and GyrB tended to
have relatively small changes in quinolone MICs (1 or 2 doubling
dilutions) compared to the parent strains and remained susceptible to
all or most of the quinolones tested. However, some mutants with no
mutations developed resistance to some of the quinolones. For example,
strains 3, 4, 5, 9, and 10 exposed to ciprofloxacin developed
resistance to ciprofloxacin (MICs of 4 to 16 µg/ml), and strain
10 exposed to levofloxacin developed resistance to ciprofloxacin (MIC
of 16 µg/ml) and levofloxacin (MIC of 4 µg/ml).
Efflux mechanism.
Since several mutants were resistant to one
or more quinolones and did not have any mutations in ParC, ParE, GyrA,
and GyrB, we investigated the possibility that an efflux mechanism
contributed to the raised MICs. This was accomplished by determining
quinolone MICs in the absence and presence of reserpine (a known efflux pump inhibitor). MICs were determined for mutant strains for which the
MIC of a particular quinolone (after 10 subcultures on drug-free medium) was at least fourfold greater than the corresponding MIC for
the parent strain. The results are summarized in Table
3. Twenty-nine of 37 mutants had
ciprofloxacin MICs that were 2 to 16 times lower in the presence of
reserpine. Eight mutants had lower levofloxacin MICs, and one mutant
had lower trovafloxacin MICs. In all cases the levofloxacin and
trovafloxacin MICs were only 2 times lower in the presence of
reserpine. No strains had lower grepafloxacin or sparfloxacin MICs in
the presence of reserpine. Three different phenotypes among the mutants
were observed in relation to the quinolones affected by reserpine: 20 mutants had only lower ciprofloxacin MICs, 8 mutants had lower
ciprofloxacin and levofloxacin MICs, and 1 mutant had lower
ciprofloxacin and trovafloxacin MICs. Reserpine lowered the
ciprofloxacin MIC for all mutants that were resistant to ciprofloxacin
yet had no mutations in ParC, GyrA, ParE, or GyrB (strains 3, 4, 5, 9, and 10 exposed to ciprofloxacin and strain 10 exposed to levofloxacin).
Reserpine had no effect on the levofloxacin MIC for only one mutant
(strain 10 exposed to levofloxacin) that had no mutations in ParC,
GyrA, ParE, or GyrB.
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DISCUSSION |
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Materials and Methods
Results
Discussion
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The increased incidence of drug-resistant pneumococci observed in recent years may be due to selective pressure caused by drug abuse. In this study we obtained only one mutant whose amoxicillin-clavulanate MIC increased fourfold (still susceptible; MIC, 0.125 µg/ml) after serial passages in subinhibitory concentrations of amoxicillin-clavulanate. A study by Pankuch et al. (18) had similar findings. However, azithromycin readily selected for resistant mutants (18). In the present study we were readily able to select for quinolone-resistant S. pneumoniae mutants after serial passages in subinhibitory concentrations of quinolones. Among the quinolones, exposure to trovafloxacin selected for resistance in the least number of strains (8 versus 10). Additionally, trovafloxacin retained the greatest in vitro potency against mutants resistant to the other quinolones. A previous study by Gootz et al. found that trovafloxacin selected for first-step mutants less frequently than ciprofloxacin (8). Levofloxacin selected for a fourfold or greater increase in MICs for all 10 pneumococcal strains, but in contrast to the case for the other quinolones, the minimum number of subcultures required for this to occur was 14 (i.e., longer than for other quinolones). This may have therapeutic significance.
Gene sequencing of the QRDRs of parC and gyrA in
this study has shown, as in previous studies (8, 10, 12, 15,
16, 21), that the mutations associated with quinolone
resistance were Ser79Phe or Tyr and Asp83Gly or His in ParC
and Ser83Phe or Tyr and Glu87Lys in GyrA. Mutations not
previously described for S. pneumoniae that were also
associated with quinolone resistance were Asp83Ala, Asn, or Val in
ParC and Glu87Gln in GyrA. For the majority of the
quinolone-resistant mutants, resistance occurred by stepwise selection
in which low-level resistance was associated with a mutation in either
ParC or GyrA (depending on the primary target of the quinolone used as
the selecting agent) and high-level resistance was associated with
mutations in both ParC and GyrA. Gootz et al. previously reported that
mutants with double mutations in ParC and GyrA were resistant to
trovafloxacin (MIC of 4 to 16 µg/ml) (8). In this
study we observed the same trovafloxacin MICs for mutants with double
mutations when the ParC mutation was at Ser79; however, when the ParC
mutation was at another position (e.g., D83) the trovafloxacin MICs
were 1 to 2 µg/ml. Another ParC mutation not previously described
but which was not associated with resistance was Ala115Val. The fact
that Val is very similar to Ala probably accounts for the lack of
association with resistance to quinolones; however, the possibility
that Ala115 is not a critical residue for quinolone resistance also
should be considered.
Gene sequencing of the QRDRs of parE and gyrB in this study has shown, as in previous studies by Pan et al. (16) and Perichon et al. (19), that mutations at Asp435 are associated with quinolone resistance. In some cases mutations in ParE and GyrB may play a major role in quinolone resistance, as evidenced by one mutant in this study (strain 2 subcultured in levofloxacin) that had mutations at Asp435 in both ParE and GyrB and was resistant to all of the quinolones except trovafloxacin. In contrast, mutations in the QRDR of GyrB at Ser405 or Gly406 did not appear to be important in quinolone resistance, since they were not associated with any resistance.
It has been previously reported by Pan and Fisher that the primary target of ciprofloxacin is ParC (17). In this study all mutants with a mutation(s) in topoisomerase IV and/or DNA gyrase derived from subculturing in ciprofloxacin had mutations first appear in ParC. In contrast, mutants derived from subculturing in any of the other quinolones (including sparfloxacin, which Pan and Fisher [17] reported targeted GyrA) had mutations first appear in some cases in ParC and in other cases in GyrA. Interestingly, all mutants with a mutation at Asp435 in ParE and/or GyrB were selected by subculturing in levofloxacin, which suggests that ParE and GyrB may be important targets of levofloxacin. The broad-spectrum activity against pneumococci (compared to ciprofloxacin) of grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin may be due in part to their ability to act on more than one target.
As in other studies, mutations in ParC, ParE, GyrA, and GyrB were not associated with all increases in MICs, as some mutants for which MICs of quinolones were increased did not have any mutations in ParC, ParE, GyrA, and GyrB. Clearly, other mechanisms of fluoroquinolone resistance exist in S. pneumoniae. Baranova and Neyfakh (3) and Brenwald et al. (4) have recently provided evidence for a multidrug transporter that may be responsible for ciprofloxacin and norfloxacin resistance. Also, there may exist an efflux mechanism similar to NorA in Staphylococcus aureus (23). In this study we observed 29 mutants for which MICs of at least one of the quinolones were lower in the presence of reserpine, which suggests the involvement of an efflux mechanism. Additionally there may be more than one type of efflux mechanism involved, since there were three different phenotypes observed among the mutants in relation to the quinolones affected by reserpine.
The clinical significance of the findings of this study is uncertain. While we were readily able to select for quinolone-resistant S. pneumoniae mutants after serial passages in subinhibitory concentrations of quinolones, the incidence of quinolone resistance in naturally occurring strains is extremely low despite the fact that quinolones such as ciprofloxacin and ofloxacin have been used clinically for over 10 years. However, many of the same mutations in ParC and GyrA observed in the in vitro-selected quinolone-resistant mutants have been observed by us in ciprofloxacin-resistant S. pneumoniae clinical isolates (data not shown). A better understanding of the mechanisms of resistance should help to keep the levels of quinolone resistance in clinical isolates low.
This study indicates that in contrast to the case for amoxicillin-clavulanate, sequential subculture in subinhibitory concentrations of all quinolones tested led to substantially increased MICs. Oral drugs such as cephalosporins and macrolides have been shown to select for resistance in the pneumococcus (18), and the potential for this to occur with quinolones has been demonstrated in this paper. In order to help minimize the emergence of quinolone-resistant pneumococci, we feel that these findings emphasize the need for cautious and judicious use of broad-spectrum quinolones in the treatment of community-acquired respiratory tract infections.
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ACKNOWLEDGMENT |
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This study was supported by a grant from SmithKline Beecham Laboratories, Collegeville, Pa.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathology, Hershey Medical Center, 500 University Dr., Hershey, PA 17033. Phone: (717) 531-5113. Fax: (717) 531-7953. E-mail: pappelbaum{at}psghs.edu.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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