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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, November 2000, p. 3049-3054, Vol. 44, No. 11
Department of Microbiology, Mount Sinai
Hospital and Toronto Medical Laboratories, University Health
Network,1 and Department of Laboratory
Medicine and Pathobiology, University of
Toronto,2 Toronto, Ontario, Division
of Infectious Diseases, McMaster University, Henderson Site, Hamilton
Health Sciences Corporation, Hamilton,
Ontario,3 and Department of
Microbiology, Queen Elizabeth II Health Sciences Centre, Dalhousie
University, Halifax, Nova Scotia,4 Canada
Received 20 April 2000/Returned for modification 27 June
2000/Accepted 12 August 2000
We report on amino acid substitutions in the quinolone
resistance-determining region of type II topisomerases and the
prevalence of reserpine-inhibited efflux for 70 clinical isolates of
S. pneumoniae for which the
ciprofloxacin MIC is Streptococcus pneumoniae
is a leading cause of bacterial respiratory tract infections and
meningitis (3). Due to the increasing resistance of this
bacterium to commonly used antimicrobials, fluoroquinolones with
enhanced activity against gram-positive bacteria have been recommended
for the treatment of pneumococcal infections (2). Concerns
have been raised regarding the emergence of resistance to the
fluoroquinolones in S. pneumoniae, as clinical isolates with
reduced susceptibility to this class of agents have already been
reported (5, 13).
The fluoroquinolones inhibit DNA replication by forming cleavage
complexes with type II topoisomerases: topoisomerase IV and DNA
gyrase (for a review, see reference 7). In
S. pneumoniae, topoisomerase IV and DNA gyrase
are tetrameric enzymes encoded for by parC-parE and
gyrA-gyrB, respectively. Several in vitro studies have shown
that fluoroquinolone resistance in S. pneumoniae occurs in a
stepwise fashion (11, 12, 17, 27). In the case of
ciprofloxacin, low-level resistance occurs first with a mutation
in parC and progresses to higher levels with an additional mutation in gyrA (19). Specific amino acid
substitutions in the quinolone resistance-determining regions (QRDRs)
have been reported and typically include amino acid
substitutions at Ser-79 and Asp-83 in ParC and Ser-81 and Glu-85 in
GyrA (19). Although mutations in ParE and GyrB have been
reported, their contributions to resistance and in particular to
high-level resistance are unclear (6, 23, 24). In addition,
efflux, mediated by the presumed membrane protein PmrA, has also been
shown to contribute to resistance; however, its role in high-level
resistance remains uncertain (10, 29).
The knowledge of fluoroquinolone resistance in S. pneumoniae, in particular, the contribution afforded by specific
type II topoisomerase mutations, has been primarily based on in vitro selection studies and to a lesser extent on the genetic
characterization of resistant clinical isolates. Few studies have
identified large numbers of clinical isolates for which ciprofloxacin
MICs are Characterization of bacterial strains.
A total of 98 S. pneumoniae isolates were collected in 1988 and from
October 1993 to September 1998. Isolates were collected from 40 different laboratories located in eight provinces as part of a
Canada-wide surveillance program. The MICs of ciprofloxacin, sparfloxacin, levofloxacin, grepafloxacin, gatifloxacin, trovafloxacin, moxifloxacin, and gemifloxacin were determined for each isolate. The
drugs were supplied by their respective manufacturers. Susceptibility testing was performed by the broth microdilution method according to
National Committee for Clinical Laboratory Standards (NCCLS) guidelines
(18).
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Fluoroquinolone Resistance in Clinical Isolates of
Streptococcus pneumoniae: Contributions of Type II
Topoisomerase Mutations and Efflux to Levels of
Resistance
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
4 µg/ml and 28 isolates for which the ciprofloxacin MIC is 
2 µg/ml. The amino acid
substitutions in ParC conferring low-level resistance (MICs, 4 to 8 µg/ml) included Phe, Tyr, and Ala for Ser-79; Asn, Ala, Gly, Tyr, and
Val for Asp-83; Asn for Asp-78; and Pro for Ala-115. Isolates with
intermediate-level (MICs, 16 to 32 µg/ml) and high-level (MICs, 64 µg/ml) resistance harbored substitutions of Phe and Tyr for Ser-79 or
Asn and Ala for Asp-83 in ParC and an additional substitution in GyrA
which included either Glu-85-Lys (Gly) or Ser-81-Phe (Tyr).
Glu-85-Lys was found exclusively in isolates with high-level
resistance. Efflux contributed primarily to low-level resistance in
isolates with or without an amino acid substitution in ParC. The impact of amino acid substitutions in ParE was minimal, and no substitutions in GyrB were identified.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
8 µg/ml, thereby making it difficult to draw conclusions
as to what mutations or combinations thereof contribute to resistance (1, 13, 14). In our study, we characterized the QRDRs of the
type II topoisomerase genes in 98 previously reported (5) Canadian clinical isolates of S. pneumoniae for which the
ciprofloxacin MICs were 4 µg/ml (n = 70
isolates) and 2 µg/ml (n = 28 isolates). This
allowed us to examine the possible mechanisms of resistance acquired by S. pneumoniae which have
potentially been exposed to ciprofloxacin, ofloxacin, or norfloxacin.
The in vitro activities of the newer fluoroquinolones, to which these
clinical isolates had not been previously exposed, were also examined
in order to evaluate the effects of the existing type II topoisomerase mutations.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
4 µg/ml were
chosen for characterization of the mechanisms of fluoroquinolone resistance. Serotyping and pulsed-field gel electrophoresis had previously demonstrated (5) that these isolates were of
multiple clones and serotypes, including serotypes 11A, 23F, 9V, 6A,
6B, 9N, 22F, and 14. For comparative purposes, a random sample of 28 strains for which the ciprofloxacin MIC was 2 µg/ml was also characterized.
Isolation of DNA and PCR amplification.
Crude cell lysates
were used as DNA templates for PCR. Briefly, a loop of overnight growth
on Columbia nutrient agar supplemented with 5% sheep blood was
suspended in 100 µl of lysis buffer (100 mM NaCl, 10 mM Tris-HCl [pH
8.3], 1 mM EDTA, 1% Triton X-100) and the mixture was boiled for 10 min; 10 µl of the supernatant was used as the template in a 50-µl
reaction volume. The QRDRs of parC, parE, and
gyrA of all strains were amplified by PCR. The QRDR of
gyrB was analyzed for 21 strains for which the ciprofloxacin MIC was 16 µg/ml and for 5 strains for which the ciprofloxacin MIC
was 4 µg/ml. Primers were based on published sequences (19, 23). Amplification products were purified with either the
QIAquick PCR purification kit (Qiagen Inc., Mississauga, Ontario,
Canada) or the Concert Rapid PCR purification kit (Life Technologies, Burlington, Ontario, Canada).
Sequencing and identification of mutations. DNA sequencing was performed by ABI Prism Big Dye terminator cycle sequencing with the ABI 377 automated sequencer (PE Applied Biosystems, Mississauga, Ontario, Canada). Nucleotide and amino acid sequence comparisons were performed with the multiple-alignment sequence function of Vector NTI Suite software (InforMax Inc., Bethesda, Md.). The GenBank accession numbers for the wild-type sequences used for comparison purposes were Z67739 for parC and parE (20), AB010387 for gyrA and Z67740 for gyrB (16).
Active efflux.
Isolates for which ciprofloxacin MICs were
2 µg/ml were examined for active efflux by agar dilution on
Mueller-Hinton agar containing 5% sheep blood in the presence of
ciprofloxacin with or without 10 mg of the alkaloid reserpine (Sigma
Chemical Co., St. Louis, Mo.) per ml (4). Strains for which
there was a fourfold or greater decrease in the ciprofloxacin MIC in
the presence of reserpine were considered in this study to be positive
for reserpine-inhibited efflux.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Bacterial strains.
For the purposes of this study, the
isolates were categorized according to the ciprofloxacin MIC. Six
isolates were classified as having high-level resistance (MIC, 64 µg/ml), 15 were classified as having intermediate-level resistance
(MICs, 16 to 32 µg/ml), 49 were classified as having low-level
resistance (MICs, 4 to 8 µg/ml), and 28 were classified as
susceptible (MIC, 2 µg/ml).
Type II topoisomerase amino acid substitutions and levels of
resistance.
All isolates with high- and intermediate-level
resistance had at least one amino acid substitution in ParC and all but
one, an isolate with intermediate-level resistance, had an additional substitution in GyrA. In contrast, only 6 of the 49 (12%) strains with
low-level resistance had amino acid substitutions in both ParC and GyrA
(Fig. 1). Amino acid substitutions in
ParC, but not in GyrA, were identified in 35 of the 49 (71%) isolates
with low-level resistance and 12 of 28 (43%) susceptible isolates. Strains lacking ParC and GyrA substitutions were found only among isolates for which the ciprofloxacin MICs were 4 µg/ml (8 of 49 [16%] and 6 of 8 [57%] for the low-level resistance and
susceptible groups, respectively) (Fig. 1). ParE
substitutions were detected in 4 of the 6 (67%) isolates with
high-level resistance isolates, 13 of the 15 (87%) isolates with
intermediate-level resistance, 33 of the 49 (67%) isolates
with low-level resistance, and 20 of the 28 (71%) susceptible
isolates. In the case of GyrB, no amino acid substitutions in the QRDR
were detected for any of the 26 strains tested (the ciprofloxacin MICs
for the isolates characterized ranged from 4 to 64 µg/ml).
|
Specific amino acid substitutions in QRDRs of ParC and GyrA for
isolates with intermediate- and high-level resistance (MICs, 16
µg/ml).
All 21 isolates for which the ciprofloxacin MIC was 16
µg/ml had at least one substitution in the QRDR of ParC. Amino acid substitutions within this group were detected at positions 52, 79, 83, and 137 (Table 1). However, 18 of these
isolates (86%) had a substitution at Ser-79 to either Phe or Tyr. The
Ser-79-Phe substitution was more prevalent, with 5 of the 6 (83%)
isolates with high-level resistance and 8 of the 15 (53%) isolates
with intermediate-level resistance harboring this substitution. Other less frequent substitutions were also detected in both groups, including Ser-52-Gly (two isolates with high-level resistance), Lys-137-Asn (three and two isolates with high- and intermediate-level resistance, respectively), Asp-83-Asn (two isolates with
intermediate-level resistance), and Asp-83-Ala (one isolate with
intermediate-level resistance) (Table 1).
|
32 µg/ml and in all but one of the isolates for which the ciprofloxacin MIC was 16 µg/ml (Table 1). Unlike ParC, the amino acid substitutions in GyrA occurred at only two amino acid positions (Ser-81 and Glu-85). Thirteen of the 20 isolates with GyrA substitutions (11 of the 14 [79%] isolates with
intermediate-level resistance and 2 of the 6 [33%] isolates with
high-level resistance) harbored a Phe substitution at position 81, and
only 2 of the 14 (14%) isolates with intermediate-level
resistance had a Ser-81-Tyr substitution. Interestingly, only
strains with high-level resistance harbored the Glu-85-Lys substitution
(four of the six [67%] isolates). Although a substitution at
Glu-85 was noted in one isolate for which the ciprofloxacin MIC was 16 µg/ml, the amino acid change was to Gly and not Lys.
Specific amino acid substitutions in QRDRs of ParC and GyrA for low-level resistance (MICs, 4 to 8 µg/ml). Twenty-five of the 41 (61%) isolates with an amino acid substitution in ParC harbored a substitution for Ser-79 (Table 1). Of these isolates, 18 (44%) had Ser-79-Phe substitutions and 6 (15%) had Ser-79-Tyr substitutions. Substitutions were also detected at positions Ser-52, Asp-83, and Lys-137. However, unlike the isolates with intermediate- and high-level resistance, the following substitutions were also noted: Ser-79-Ala, Asp-83-Gly, Asp-83-Val, Asp-83-Tyr, Ala-115-Pro, and Asp-78-Asn (Table 1).
Although GyrA substitutions were detected in only a small proportion of the isolates for which the ciprofloxacin MIC was 4 to 8 µg/ml (6 of the 49 [12%] isolates) compared with the proportions of isolates with high- and intermediate-level resistance with GyrA substitutions, identical amino acid substitutions were noted. These included Ser-81-Phe (five of six [83%] isolates) and Glu-85-Gly (one of six [17%] isolates) (Table 1).Specific amino acid substitutions in QRDRs of ParC and GyrA for
susceptible isolates (MICs, 2 µg/ml).
Twelve of the 28 (43%)
susceptible isolates had a substitution for residues in the QRDR of
ParC (Table 1). Of these, for nine the ciprofloxacin MIC was 2 µg/ml,
for one the ciprofloxacin MIC was 1 µg/ml, and for two the
ciprofloxacin MIC was 0.5 µg/ml. Mutations in parC
resulting in amino acid substitutions of Ser-79-Phe (3 [25%]
isolates) or Ser-79-Tyr (two [17%] isolates) were detected only in
isolates for which ciprofloxacin MICs were 2 µg/ml. Other substitutions included Lys-137-Asn (six [50%] isolates), Ser-52-Gly (one [8%] isolate), and Tyr-129-Ser (one [8%] isolate). Of the isolates for which the ciprofloxacin MIC was 1 µg/ml, only
Ser-52-Gly and Lys-137-Asn substitutions were noted.
Specific amino acid substitutions in QRDRs of GyrB and ParE.
Amino acid substitutions in ParE were detected in 67% of the isolates
with high-level resistance, 87% of the isolates with intermediate-level resistance, 67% of the isolates with low-level resistance, and 71% of the susceptible strains (Table 1). In particular, four of nine isolates (44%) for which the ciprofloxacin MIC was 0.5 µg/ml had ParE substitutions. With the exception of two
isolates with an Ala-468-Val substitution (ciprofloxacin MICs, 0.25 and
2 µg/ml, respectively), all strains harboring a ParE substitution had
an Ile-to-Val substitution at position 460. In addition, Arg-447-Ser
and Asp-435-Asn substitutions were also found in combination with the
Ile-460-Val substitution in two isolates with low-level resistance. No
amino acid substitutions in the QRDR of GyrB were detected in any of
the 26 strains tested (21 isolates for which the ciprofloxacin MICs
were 16 µg/ml and 5 isolates for which the ciprofloxacin MIC was 4 µg/ml).
Comparative ciprofloxacin susceptibilities and specific
ParC-GyrA amino acid substitution combinations.
In order
to determine if particular amino acid substitution combinations
in the QRDRs would correlate with particular ciprofloxacin MICs,
all 98 isolates were grouped according to their ParC-GyrA substitutions
together with the MIC range (Table 2). Of
the 13 isolates which harbored a substitution for Asp-83, only 4 harbored an additional substitution in GyrA and the ciprofloxacin MIC
range never exceeded 16 µg/ml. In contrast, for isolates harboring an identical GyrA substitution but in combination with a substitution for
Ser-79 instead of Asp-83, ciprofloxacin MICs were as high as 64 µg/ml. Ciprofloxacin MICs for isolates harboring a substitution in
ParC (for either Ser-79 or Asp-83) but not in GyrA ranged from 0.5 to
16 µg/ml; however, of these isolates the MIC was 16 µg/ml for only
one isolate, and that isolate was also positive for reserpine-inhibited efflux. Substitutions of Asn for Lys-137 and Gly for Ser-52 in isolates
for which the ciprofloxacin MIC was 16 µg/ml were found only in
combination with an additional substitution at either Ser-79 or Asp-83.
For eight isolates which harbored only the Lys-137-Asn substitution and
no GyrA substitution, the ciprofloxacin MICs were in the range of 0.5 to 4 µg/ml. Incidentally, for four of these eight isolates the
ciprofloxacin MIC was 4 µg/ml, but the isolates were also classified
as positive for reserpine-inhibited efflux.
|
Active efflux phenotype.
Active efflux, which was evaluated
indirectly by reserpine reduction of the ciprofloxacin MIC, was tested
for all isolates for which the ciprofloxacin MIC was 2 µg/ml. As
shown in Fig. 1, all strains for which the ciprofloxacin MIC was 32
µg/ml were found to be negative for reserpine-inhibited efflux and
only 1 of the 11 strains with intermediate-level resistance for which the ciprofloxacin MIC was 16 µg/ml was positive for
reserpine-inhibited efflux. In fact, a 16-fold decrease in the
ciprofloxacin MIC was noted for this strain when it was grown in the
presence of reserpine. Interestingly, this strain with
intermediate-level resistance had amino acid substitutions only in ParC
and not in GyrA. In contrast, 18 of the 49 (37%) isolates with
low-level resistance (MICs, 4 to 8 µg/ml) were positive for
reserpine-inhibited efflux (Fig. 1). Moreover, of these 18 strains, 8 isolates for which the ciprofloxacin MIC was 4 µg/ml had no
substitutions in either ParC or GyrA. Only 3 of the 28 (11%)
susceptible strains (ciprofloxacin MIC, 2 µg/ml) were positive for
reserpine-inhibited efflux.
Comparative fluoroquinolone susceptibilities and their association
with amino acid substitutions in ParC and GyrA.
Table
3 shows that of the other
fluoroquinolones tested, all displayed improved activity against
S. pneumoniae relative to the activity of ciprofloxacin.
However, for isolates harboring both a ParC and GyrA substitution the
MICs of these fluoroquinolones were higher than those for isolates with
a substitution in ParC only. One isolate harbored neither a ParC nor a
GyrA mutation, but the gemifloxacin MIC for the isolate was 0.25 µg/ml and the isolate had an amino acid substitution of Ser for
Arg-447 in ParE (Table 3). This is in contrast to the nine additional
isolates for which the gemifloxacin MIC was 0.25 µg/ml, which had
amino acid substitutions in the QRDRs of both ParC and GyrA but did not
harbor the Arg-447-Ser substitution in the QRDR of ParE.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Our study provides a detailed analysis of the type II topoisomerase mutations that arise in clinical isolates that potentially have been exposed only to quinolones that preferentially target topoisomerase IV and in particular the ParC subunits in S. pneumoniae. In accordance with the predicted stepwise generation of mutations that confer resistance, none of the isolates characterized harbored DNA gyrase QRDR mutations in the absence of topoisomerase IV QRDR mutations. Consistent also with in vitro selection studies (11, 12, 17, 19, 27) was our finding that low-level resistance typically arises following an amino acid substitution for Ser-79 or Asp-83 in ParC and that intermediate- and high-level resistance is achieved following a subsequent substitution for Ser-81 or Glu-85 in GyrA. Furthermore, our findings suggest that the acquisition of a GyrA substitution, in combination with a ParC substitution, is sufficient to increase the MICs of fluoroquinolones that are more active than ciprofloxacin against gram-positive organisms.
Of the ParC substitutions associated with resistance in this study (Ser-79-[Phe, Tyr, Ala], Asp-83-[Ala, Asn, Gly, Tyr, Val], Asp-78-Asn, and Ala-115-Pro), all but the substitutions of Ala for Ser-79 and Pro for Ala-115 have previously been reported for S. pneumoniae (9, 13, 19, 21, 22, 24-26, 28). In addition, we also found amino acid substitutions such as Lys-137-Asn (13, 17, 26) and Ser-52-Gly which were not associated with increased MICs. Of note was the finding that the substitutions of Ser-79-Ala, Asp-83-(Gly, Tyr, Val), Asp-78-Asn, and Ala-115-Pro were associated only with low-level resistance. These findings suggest that both the location of the ParC residue mutated and the specific amino acid substituted for that residue determine whether an isolate has the potential of attaining high levels of resistance. For example, isolates with the ParC amino acid substitutions of Asp-78-Asn and Ala-115-Pro exhibited only low-level resistance, even though they had QRDR substitutions in GyrA identical to those found in isolates with intermediate- and high-level resistance. In addition, the likelihood of Ser(TCT)-79-Ala(GCT) ever being converted to the more critical substitution of Ser(TCT)-79-Phe(TTT) is rare since a double nucleotide change would be required, thereby suggesting that the amino acid substitution in ParC determines the level of resistance attainable by a particular isolate.
In contrast to the heterogeneity of the amino acid substitutions in
ParC, GyrA substitutions occurred at only two amino acid positions:
Ser-81-(Phe, Tyr) and Glu-85-(Lys, Gly) (19, 21, 22, 26,
28). The substitution of Lys for Glu-85 in GyrA was identified
only in isolates with high-level resistance, while its substitution to
Gly was associated only with intermediate-level resistance
(ciprofloxacin MIC, 16 µg/ml). The equivalent residue in
Escherichia coli forms part of the 
-helix in GyrA, which
lies adjacent to what is thought to be the active site for DNA
breakage-reunion reactions and the site for fluoroquinolone binding
(15). We speculate that the bulkier side chain of the Lys
residue, in contrast to the hydrogen atom of Gly, would significantly
reduce quinolone-binding affinity by steric hindrance. Moreover, the
positively charged amino group of the Lys side chain at this position
may also reduce the binding affinity and hence the effectiveness of the fluoroquinolone.
The significance of the substitutions identified in ParE was less clear than the significance of those identified in ParC and GyrA. A previously reported substitution of Asp-435-Asn in ParE (23, 24) was observed in this study in two isolates with low-level resistance; however, neither isolate harbored the conventional QRDR ParC amino acid substitution, thereby suggesting that Asp-435 may be important for quinolone-topoisomerase IV-DNA binding. Moreover, it also demonstrates that while amino acid substitutions generally occur first in ParC, the drug recognizes not just a single subunit but, rather, the topoisomerase IV complex as a whole (8). In contrast to Asp-435-Asn, the substitution of Val for Ile-460, as described previously (24), appears to play no role in resistance, as it was identified at all levels of resistance and in similar percentages of isolates. In addition, the substitution of Ser for Arg-447 was associated with an elevated gemifloxacin MIC (0.25 µg/ml) compared to those for isolates with no documented mutations. This particular isolate did not have a substitution in the QRDR of either ParC or GyrA, thereby suggesting that this substitution may result in the decreased activity of gemifloxacin against S. pneumoniae.
Our findings suggest that reserpine-inhibited efflux contributes
primarily to lower levels of ciprofloxacin resistance and may do so
either alone or to a lesser extent by complementing QRDR substitutions
in ParC. Interestingly, of the isolates with low-level resistance
identified that demonstrated reserpine-inhibited efflux with ParC
substitutions, the majority harbored amino acid substitutions not
associated with resistance (Ser-52-Gly and Lys-137-Asn) or
substitutions that are associated exclusively with low-resistance (Asp-83-[Gly, Tyr]). No isolates with both reserpine-inhibited efflux
and a gyrA mutation were identified. Although it is tempting to speculate that efflux is down-regulated following the acquisition of
an amino acid substitution in the QRDR of GyrA, it is also possible
that the doubling dilution technique used in this study was an
inaccurate measure of efflux in isolates for which the ciprofloxacin
MICs were 32 µg/ml. Although further studies are needed to support
the hypothesis of down-regulation, it would seem likely that an
organism that has achieved resistance to a particular drug by
chromosomal mutations would not require such an energetically costly process.
Our study supports the predicted stepwise generation of mutations in S. pneumoniae. For the most part, isolates for which the ciprofloxacin MIC was greater than or equal to 16 µg/ml have both ParC and GyrA substitutions of Ser-79 or Asp-83 and Ser-81 or Glu-85, respectively. However, ciprofloxacin MICs cannot always be predicted for isolates with given QRDR amino acid substitutions. For example, the ciprofloxacin MICs for two isolates harboring identical QRDR mutations in ParC and GyrA had a fourfold difference. This suggests the presence of unidentified mutations in regions outside the currently recognized QRDR and/or additional mechanisms yet to be identified.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the Canadian Bacterial Diseases Network. D.J.B. is a recipient of a Medical Research Council of Canada postdoctoral research fellowship.
We thank Tiffany Tam and Heather MacKenzie for help with the PCR.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Rm 1483, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario, Canada M5G 1X5. Phone: (416) 586-8459. Fax: (416) 586-8746. E-mail: jdeazavedo{at}mtsinai.on.ca.
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Abstract
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Materials and Methods
Results
Discussion
References
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