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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, February 2000, p. 462-466, Vol. 44, No. 2
MRL Pharmaceutical Services, Utrecht, The
Netherlands1; MRL Pharmaceutical
Services, Herndon, Virginia2; and
Institute for Medical Microbiology and Virology,
Heinrich-Heine University Düsseldorf,
Düsseldorf,3 and Institute of
Pharmaceutical Microbiology, University of Bonn,
Bonn,4 Germany
Received 2 July 1999/Returned for modification 3 September
1999/Accepted 16 November 1999
From 8,419 worldwide clinical isolates of Streptococcus
pneumoniae obtained during 1997-1998, 69 isolates with reduced
susceptibility or resistance to fluoroquinolones (FQs) were molecularly
characterized. For the isolates in this prevalence study, only
parC (Ser-79 Streptococcus pneumoniae
is a leading cause of illness in humans (32). Recent
increases in resistance (4, 8, 9, 29-31) have spawned the
development of several new fluoroquinolones (FQs) with improved in
vitro antipneumococcal activity (1, 7, 10-12, 14, 15, 34,
35). In pneumococci, reports indicate mutations in
gyrA, gyrB, parC, and parE
to be associated with FQ resistance (16, 18, 20-23, 28,
33). Efflux is also reported to contribute significantly to
reduced susceptibility for some hydrophilic FQs, such as ciprofloxacin,
while more hydrophobic FQs, like grepafloxacin, appear less affected
(5, 13).
(This work was presented at the 39th Interscience Conference on
Antimicrobial Agents and Chemotherapy, 1999.)
This work aimed to define the prevalence of predominant mutations
conferring FQ resistance in pneumococci collected during 1 year.
Mutations in genes conferring FQ resistance in S. pneumoniae (16, 18, 20-23, 28, 33) have been well studied, but studies have typically included either clinical isolates (few and locally derived) or laboratory-derived mutants. In contrast, this study, the
largest molecular surveillance study of FQ resistance in S. pneumoniae to date, comprises clinically significant isolates from
locations worldwide, providing the opportunity to characterize the
prevalence of mutations globally and their impact on the MICs of
several new FQs.
A total of 8,419 clinically significant isolates of S. pneumoniae associated with lower respiratory tract or blood
infections were derived from 519 geographically distinct hospital
laboratories in Austria, People's Republic of China, France, Germany,
Italy, Japan, Spain, Switzerland, the United Kingdom, and the United States, in studies undertaken by MRL Pharmaceutical Services during 1997 and 1998 (24, 30; M. L. Hickey, C. Thornsberry, D. R. Diakun, S. V. Mani, and D. F. Sahm,
Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-20,
1998, and D. F. Sahm, I. A. Critchley, M. L. Hickey,
D. R. Diakun, S. V. Mani, and C. Thornsberry, Clin. Micro.
Infect., abstr. 110, 1999.) From these sources, 69 isolates were
selected, including 30 isolates requiring MICs above the National
Committee for Clinical Laboratory Standards susceptibility breakpoint
(19) of any of the new FQs originally tested in the initial
surveillance studies and 39 geographically unrelated isolates requiring
MICs of levofloxacin ranging from 0.25 to 2 µg/ml. Together, these
isolates provided a diverse strain set enabling the detection of
mutations conferring high-level FQ resistance, as well as genetic
changes reducing susceptibility. For each of the 69 isolates, MICs of
each drug were determined in a single central laboratory by a broth
microdilution assay according to the National Committee for Clinical
Laboratory Standards (19). Each isolate was characterized
with respect to mutations within gyrA, gyrB,
parC, and parE with prepared chromosomal DNA (2) as templates for PCR amplification of target regions and with previously defined primers (16, 21) and methods
(25).
The MIC distributions of each FQ tested are shown in Table
1. Overall, MICs of ciprofloxacin were
highest, and MICs of sitafloxacin were lowest. For purposes of
analysis, we considered sequence data in relation to MICs of
levofloxacin to comprise the least active of the new FQs. Without
exception, as is evident from Table 2,
all of the 30 levofloxacin-resistant isolates (for
which MICs were
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Prevalence of gyrA, gyrB, parC,
and parE Mutations in Clinical Isolates of
Streptococcus pneumoniae with Decreased Susceptibilities to
Different Fluoroquinolones and Originating from Worldwide
Surveillance Studies during the 1997-1998 Respiratory
Season
ABSTRACT
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Tyr) and gyrA (Ser-81Phe or
Tyr) mutations, especially in combination, were found to contribute
significantly to resistance. These mutations influenced the FQ MICs to
varying degrees, although the rank order of activity remains
independent of mutation type, with ciprofloxacin the least active,
followed by levofloxacin,
gatifloxacin/grepafloxacin/moxifloxacin/sparfloxacin/trovafloxacin, and
clinafloxacin/sitafloxacin. Efflux likely plays a crucial role in
reduced susceptibility for new hydrophilic FQs.
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4 µg/ml) had mutations within
gyrA (alone or in combination with other mutations in
gyrA or parC) encoding Ser-81Phe or Tyr. No
levofloxacin-susceptible isolates (for which MICs were 
2 µg/ml) possessed these mutations. Of the levofloxacin-resistant isolates, 22 had mutations within parC (alone or in combination with
other parC or gyrA mutations) encoding
Ser-79Phe, 3 had mutations encoding Asp-78Asn, and 2 had
mutations encoding Asp-83Asn (Table 2). Twenty-eight single or
combination mutations were found in gyrB and parE
(including alterations Ala-639Gln, Ala-538Ser, Arg-541Lys, Arg-545Asn, Ala-639Gln in GyrB; Glu-407Lys,
Lys-466Met, Ile-460Val, Asp-435Asn, Ile-460Val, and
Pro-454Ser in ParE). Compared to wild-type strains or strains with
single or combinational mutations in gyrA or parC
alone, with or without with these additional mutations in
gyrB and parE, none was obviously associated with
reduced susceptibility to any of the FQs, including ciprofloxacin or
levofloxacin (although complementation studies would be necessary to
confirm this as well as a comparative molecular analysis of fully
susceptible isolates). Although some authors have described a possible
role for a parE mutation in resistance (Asp-435Asn)
(17, 23) we and others have not been able to assign
significance to parE (20) or gyrB
(18) mutations.
TABLE 1.
Number of isolates inhibited at
different concentrationsa
TABLE 2.
Amino acid changes encoded by mutations in the
gyrA and parC gene loci and corresponding
fluoroquinolone MICs (mg/litera)
The impact of well-characterized alterations in both laboratory mutants
and clinical isolates, namely, Ser-81Phe or Tyr in GyrA and
Ser-79Phe in ParC, previously described by other authors (16, 18, 20-22, 28, 33), was apparent (Table 2). Other alterations previously suggested as important, including Glu-85Lys (laboratory mutant and clinical isolate) (21, 27, 33) or Trp-93Arg (clinical isolate) (27) in GyrA and
Ser-80Pro (17) (clinical isolate) in ParC, were not
found. While detected, alterations Arg-95Cys (21) and
Lys-137Asn (27) in ParC seemed not to be
significant. Thus, we conclude that such mutations are clinically rare
or not obviously associated with FQ resistance. Asp-78Asn and
Asp-83Asn alterations in ParC were only found in three and two
isolates, respectively, and their contributions to FQ resistance were
either negligible or masked, since they only occurred with a
Ser-81Phe alteration in GyrA. No previously unreported
parC, parE, gyrA, or gyrB
mutations significantly conferring reduced susceptibility to FQs were
found. Thus only classical mutations, such as those in parC
(Ser-79Phe) and gyrA (Ser-81Phe or Tyr), seem to play
a significant role in FQ resistance in this worldwide sample of
clinical S. pneumoniae isolates. Single significant mutations in parC or gyrA appeared to have
moderate effects (approximately 2 dilution increases) on MICs, similar
for each drug, although the high levels of activity of sitafloxacin and
clinafloxacin reduced this effect.
These data underscore the probable impact of efflux on FQ
susceptibility and the biovariation among strains observed when studying a diverse collection of clinical isolates in contrast to
laboratory mutants. This is exemplified by the fact that many isolates
with significant mutation(s) require MICs overlapping those for
wild-type isolates (see Table 2). This overlap is most apparent with
MICs required by isolates possessing single alterations of Arg-95Cys
or Lys-137Asn in ParC, demonstrating the minimal impact of
these mutations on susceptibility. It is especially noticeable when
considering MICs of ciprofloxacin and sparfloxacin for isolates with
multiple mutations in parC and gyrA. These
quinolones comprise the most hydrophilic of the compounds tested and
are readily effluxed; thus, higher MICs for isolates wild type at gyrA and parC loci can be observed. In contrast,
hydrophobic compounds such as gatifloxacin, grepafloxacin, and
moxifloxacin are less affected by efflux; thus, predictably, little or
no MIC overlap occurs between isolates wild type at the topoisomerase
and gyrase genes and those with detectable mutations in these loci.
One-fold-dilution overlaps are observed for some mutational
combinations for hydrophobic clinafloxacin and sitafloxacin, which can
probably be explained by the extremely low MICs of these compounds and
the reduced impact of mutational events on activity. These results are
similar to data derived previously for efflux studies in
Staphylococcus aureus (26).
The order of activity of drugs (Table 1 and 2) is generally conserved throughout, regardless of mutation(s) identified in gyrA and parC. Thus for all combinations of mutations detected, a left-to-right upward trend is evident (Table 2), with sitafloxacin as the most active compound and ciprofloxacin as the least active.
The results of this molecular epidemiological survey provide an opportunity to view the predominant mutations conferring reduced susceptibility to FQs in recent clinical pneumococcal isolates. Our findings indicate that researchers likely have characterized most of the mutations important in conferring reduced susceptibility to older FQ compounds, such as ciprofloxacin. Clearly, these mutations do impact susceptibilities to even the most active new FQs to some extent, although this varies between strains and for each drug. Based on the range of MICs of FQs for wild-type isolates, it is predicted that efflux will play a significant role for some drugs and warrants further study or that other systems have a hitherto-unidentified impact on FQ susceptibility. It will be interesting to witness the effect of selective pressures imposed on these genetic systems by the increased use of the new FQ compounds described in this study, many of which retain high levels of in vitro activity despite the presence of significant mutations in topoisomerase- and gyrase-encoding genes. This is particularly significant in light of recent work by Chen et al. (6), who report an increasing prevalence of pneumococcal resistance to fluoroquinolones. Future prevalence studies will be able to track changes in the predominant mutations conferring resistance to FQs.
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ACKNOWLEDGMENTS |
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This study was supported by a grant from Glaxo Wellcome (Greenford, United Kingdom).
Isolates included in this study were derived from surveillance studies funded by Bayer Pharmaceuticals (Groton, Conn.) and Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan). We thank Geriann Piazza for copy editing.
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FOOTNOTES |
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* Corresponding author. Mailing address: MRL Pharmaceutical Services, Den Brielstraat 11, 3554XD, Utrecht, The Netherlands. Phone: 31 30 265 1794. Fax: 31 30 265 1784. E-mail: mjones{at}thetsn.com.
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