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Antimicrobial Agents and Chemotherapy, October 1999, p. 2513-2516, Vol. 43, No. 10
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Association of Alterations in ParC and GyrA
Proteins with Resistance of Clinical Isolates of Enterococcus
faecium to Nine Different Fluoroquinolones
Sylvain
Brisse,1,*
Ad C.
Fluit,1
Ulrich
Wagner,2
Peter
Heisig,3
Dana
Milatovic,1
Jan
Verhoef,1
Sybille
Scheuring,2
Karl
Köhrer,2 and
Franz-Josef
Schmitz1,2
Eijkman-Winkler Institute, Utrecht
University, 3584 CX, Utrecht, The Netherlands,1
and Institute for Medical Microbiology and Virology, University
Hospital Düsseldorf, D-40225
Düsseldorf,2 and Institute for
Pharmaceutical Microbiology, University of Bonn,
Bonn,3 Germany
Received 30 March 1999/Returned for modification 28 June
1999/Accepted 27 July 1999
 |
ABSTRACT |
The parC and gyrA genes of 73 ciprofloxacin-resistant and 6 ciprofloxacin-susceptible
Enterococcus faecium clinical isolates were partly
sequenced. Alterations in ParC and GyrA, possibly in combination with
other resistance mechanisms, severely restricted the in vitro
activities of the nine quinolones tested. For all isolates,
clinafloxacin and sitafloxacin showed the best activities.
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TEXT |
The prevalence of infections due to
Enterococcus faecium species has been increasing over the
last few years (3, 16, 18). Until now, the available
fluoroquinolones have been of limited value in the treatment of
enterococcal infections because of their poor efficacy (25)
and the emergence of acquired fluoroquinolone resistance
(9). However, new drugs, such as clinafloxacin and sitafloxacin, are showing increased activity against enterococci (1, 13, 15, 29, 31).
The purpose of the study described here was to characterize the
parC and gyrA genes of 73 ciprofloxacin-resistant
and 6 ciprofloxacin-susceptible isolates of E. faecium. In
addition, the in vitro activities of nine fluoroquinolones were
compared (see Table 1).
The isolates tested originated from 24 European university hospitals
participating in the European SENTRY Antimicrobial Surveillance Program
(8, 27). Between April 1997 and December 1998, 116 epidemiologically nonrelated E. faecium isolates were
collected. Of these, 94 (81%) were intermediate or fully resistant to
ciprofloxacin. An epidemiologically representative subset of 73 ciprofloxacin-resistant isolates and 6 ciprofloxacin-susceptible
isolates was analyzed.
MICs were measured by using concentrations of antibiotics that ranged
from 0.06 to 512 µg/ml and were determined by a broth microdilution
method (19).
The gyrA and parC portions of the genomes of the
E. faecium isolates, which are homologous to the quinolone
resistance-determining region of Escherichia coli
(30), were amplified and sequenced as described previously
(26). The primers were designed according to the
gyrA and parC sequences of E. faecium
(EMBL database accession nos. AF060881 and AB017811, respectively).
Primers gyrA-A (5'-CGGCGGCACCGTCACCGTCAACAG-3';
nucleotides [nt] 139 to 162), gyrA-C
(5'-GAATTGGGTGTGACACCGGATAAAG-3'; nt 579 to 558),
parC-A (5'-TTCCCGTGCATTTCGATCAGTACTTC-3'; nt 185 to 204), and parC-C (5'-CGTATGACAAAGGATTCCGTAAATC-3';
nt 573 to 554) were used.
Sequences with no mutations were defined as being identical to the
reference EMBL sequences. The alterations found in E. faecium GyrA and ParC proteins are indicated in Table
1.
View this table:
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TABLE 1.
Amino acid changes within ParC and GyrA in 79 E. faecium isolates and corresponding MICs of 9 fluoroquinolones
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Eight different single or combined amino acid changes were detected in
42 of the 73 ciprofloxacin-resistant isolates of E. faecium.
No amino acid change was detected in either ParC or GyrA of the
remaining 31 ciprofloxacin-resistant isolates or the 6 susceptible isolates.
Thirty-two of the ciprofloxacin-resistant E. faecium
isolates demonstrated mutations in parC, leading to an amino
acid change from Ser-80 to Ile or Arg, and 10 showed a deduced amino
acid change from Glu-84 to Lys or Thr. Thirty-six of the
ciprofloxacin-resistant isolates showed an amino acid change in GyrA,
either from Ser-83 to Arg or Leu (14 isolates) or from Glu-87 to Leu or
Gly (22 isolates). Six isolates had amino acid changes in ParC alone,
without an additional change in GyrA.
The MICs of each quinolone tested are shown in Table 1 for all isolates
with alterations in ParC and/or GyrA. These results demonstrate an
association between protein alterations and increased MICs. Indeed, for
isolates with no identifiable mutations, MICs were lower than those for
isolates with only a single amino acid change in ParC. Finally, for
those isolates with one alteration in ParC and at least one alteration
in GyrA, the highest MICs observed were, for all quinolones tested,
only 1 or 2 dilutions higher than those for isolates with only one ParC alteration.
Since ciprofloxacin is still the most commonly used quinolone, our
finding of six isolates with alterations only in ParC, together with
the fact that no isolate with just GyrA alterations was found, suggests
that topoisomerase IV is the primary target of ciprofloxacin in
E. faecium. This is similar to earlier findings for
Staphylococcus aureus (4) and Streptococcus
pneumoniae (17, 21) and therefore supports the theory
that topoisomerase IV is the primary target of ciprofloxacin in most
gram-positive bacteria (14). Moreover, our data indicate a
limited impact of additional GyrA alterations on ciprofloxacin
resistance in E. faecium, in contrast to their proposed
importance in S. aureus and S. pneumoniae
(4, 7, 14, 26).
It is known for many gram-positive pathogens, including the closely
related species Enterococcus faecalis (11), that
ciprofloxacin selects for mutants with alterations in ParC before it
selects for those with alterations in GyrA. Therefore, it is not
surprising that the ciprofloxacin-resistant clinical isolates of
E. faecium described here are either parC or
parC-gyrA mutants and not gyrA mutants. There is
evidence that gyrase is the primary target of some quinolones, such as
gatifloxacin, sparfloxacin, and clinafloxacin in Streptococcus
pneumoniae (5, 23) and sparfloxacin in Mycoplasma hominis (2). Thus, and on the basis of the fact that we
have analyzed only clinical isolates and no in vitro mutants, our
results cannot be interpreted as indicating that topoisomerase IV is
the primary target of all quinolones in E. faecium.
High-level resistance (MIC of ciprofloxacin, >16 µg/ml) in E. faecalis has been associated with either a single mutation in ParC
or combined mutations in ParC and GyrA (11). Concordantly, Korten et al. (12) and Tankovic et al. (28) found
alterations in GyrA only in high-level ciprofloxacin-resistant strains.
In the present study, we also found that all isolates with alterations in GyrA and/or ParC showed high-level resistance. However, even without
any alterations in the ParC and GyrA proteins, 25 of 31 isolates showed
either intermediate susceptibility or low-level ciprofloxacin
resistance. As noted previously (28), this suggests the
contribution of mutations in other genes. Since we have not examined
the GyrB and ParE subunits, it cannot be excluded that some isolates
have alterations in these proteins (22-24). Furthermore, active efflux of quinolones has been demonstrated in other
gram-positive cocci such as S. aureus (10, 20)
and S. pneumoniae (6).
On the basis of a breakpoint of >1 µg/ml, 2 of 79 isolates were
susceptible to grepafloxacin, 6 were susceptible to ciprofloxacin and
levofloxacin, 8 were susceptible to gatifloxacin, 11 were susceptible
to moxifloxacin, 18 were susceptible to trovafloxacin, 27 were
susceptible to sparfloxacin, 35 were susceptible to clinafloxacin, and
36 were susceptible to sitafloxacin. Moreover, sitafloxacin and
clinafloxacin showed the best in vitro activities against all isolates.
These results echo the improved activities, as reported previously
against genetically undefined isolates of E. faecium, of sitafloxacin (12, 29) and clinafloxacin (1, 16,
31). However, our study also indicates that alterations in ParC
and GyrA severely restrict their in vitro activities. These two new quinolones therefore may be of clinical value only for the treatment of
infections caused by E. faecium without alterations in ParC and GyrA proteins.
| |
ACKNOWLEDGMENTS |
We thank Marita Hautvast, Mirjam Klootwijk, Karlijn Kusters, and
Stefan de Vaal for expert technical assistance. We thank the following
members of the SENTRY Antimicrobial Surveillance Program for referring
isolates from their institutes for use in this study: Helmut
Mittermayer, Marc Struelens, Jacques Acar, Vincent Jarlier, Jerome
Etienne, Rene Courcol, Franz Daschner, Ulrich Hadding, Nikos Legakis,
Gian-Carlo Schito, Carlo Mancini, Piotr Heczko, Waleria Hyrniewicz,
Professor Dario Costa, Evilio Perea, Fernando Baquero, Rogelio Martin
Alvarez, Jacques Bille, Gary French, Nathan Keller, Volkan Korten,
Deniz Gür, and Serhat Unal.
Sylvain Brisse was supported by a European Human Capital Mobility
Grant. This work was funded in part by Bristol-Myers Squibb Pharmaceuticals via the SENTRY Antimicrobial Surveillance Program.
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FOOTNOTES |
*
Corresponding author. Mailing address: Eijkman-Winkler
Institute, Utrecht University, AZU G04.614, Heidelberglaan 100, 3585 CX, Utrecht, The Netherlands. Phone: 31 30 250-7625. Fax: 31 30 254-1770. E-mail: sbrisse{at}lab.azu.nl.
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REFERENCES |
| 1.
|
Bauernfeind, A.
1997.
Comparison of the antibacterial activities of the quinolones Bay 12-8039, gatifloxacin (AM 1155), trovafloxacin, clinafloxacin, levofloxacin and ciprofloxacin.
J. Antimicrob. Chemother.
40:639-651[Abstract/Free Full Text].
|
| 2.
|
Bebear, C. M.,
H. Renaudin,
A. Charron,
J. M. Bove,
C. Bebear, and J. Renaudin.
1998.
Alterations in topoisomerase IV and DNA gyrase in quinolone-resistant mutants of Mycoplasma hominis obtained in vitro.
Antimicrob. Agents Chemother.
42:2304-2311[Abstract/Free Full Text].
|
| 3.
|
Eliopoulos, G. M.
1993.
Increasing problems in the therapy of enterococcal infections.
Eur. J. Clin. Microbiol. Infect. Dis.
12:409-412[Medline].
|
| 4.
|
Ferrero, L.,
B. Cameron, and J. Crouzet.
1995.
Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus.
Antimicrob. Agents Chemother.
39:1554-1558[Abstract].
|
| 5.
|
Fukuda, H., and K. Hiramatsu.
1999.
Primary targets of fluoroquinolones in Streptococcus pneumoniae.
Antimicrob. Agents Chemother.
43:410-412[Abstract/Free Full Text].
|
| 6.
|
Gill, M. J.,
N. P. Brenwald, and R. Wise.
1999.
Identification of an efflux pump gene, pmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniae.
Antimicrob. Agents Chemother.
43:187-189[Abstract/Free Full Text].
|
| 7.
|
Hooper, D. C.
1995.
Bacterial resistance to fluoroquinolones: mechanisms and patterns.
Adv. Exp. Med. Biol.
390:49-57[Medline].
|
| 8.
|
Jones, M. E.,
R. N. Jones,
H. Sader,
J. Verhoef, and J. Acar.
1998.
Current susceptibilities of staphylococci to glycopeptides determined as part of an international resistance surveillance programme. Sentry Antimicrobial Surveillance Program.
J. Antimicrob. Chemother.
42:119-121[Free Full Text].
|
| 9.
|
Jones, R. N.,
E. N. Kehrberg,
M. E. Erwin,
S. C. Anderson, and the Fluoroquinolone Resistance Surveillance Group.
1994.
Prevalence of important pathogens and antimicrobial activity of parenteral drugs at numerous medical centers in the United States.
Diagn. Microbiol. Infect. Dis.
19:203-215[Medline].
|
| 10.
|
Kaatz, G. W., and S. M. Seo.
1997.
Mechanisms of fluoroquinolone resistance in genetically related strains of Staphylococcus aureus.
Antimicrob. Agents Chemother.
41:2733-2737[Abstract].
|
| 11.
|
Kanematsu, E.,
T. Deguchi,
M. Yasuda,
T. Kawamura,
Y. Nishino, and Y. Kawada.
1998.
Alterations in the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV associated with quinolone resistance in Enterococcus faecalis.
Antimicrob. Agents Chemother.
42:433-435[Abstract/Free Full Text].
|
| 12.
|
Korten, V.,
W. M. Huang, and B. E. Murray.
1994.
Analysis by PCR and direct DNA sequencing of gyrA mutations associated with fluoroquinolone resistance in Enterococcus faecalis.
Antimicrob. Agents Chemother.
38:2091-2094[Abstract/Free Full Text].
|
| 13.
|
Korten, V.,
J. F. Tomayko, and B. E. Murray.
1994.
Comparative in vitro activity of DU-6859a, a new fluoroquinolone agent, against gram-positive cocci.
Antimicrob. Agents Chemother.
38:611-615[Abstract/Free Full Text].
|
| 14.
|
Martinez, J. L.,
A. Alonso,
J. M. Gomez-Gomez, and F. Baquero.
1998.
Quinolone resistance by mutations in chromosomal gyrase genes. Just the tip of the iceberg?
J. Antimicrob. Chemother.
42:683-688[Free Full Text].
|
| 15.
|
Mercier, R. C.,
S. R. Penzak, and M. J. Rybak.
1997.
In vitro activities of an investigational quinolone, glycylcycline, glycopeptide, streptogramin, and oxazolidinone tested alone and in combinations against vancomycin-resistant Enterococcus faecium.
Antimicrob. Agents Chemother.
41:2573-2575[Abstract].
|
| 16.
|
Moellering, R. C., Jr.
1991.
The Garrod Lecture. The enterococcus: a classic example of the impact of antimicrobial resistance on therapeutic options.
J. Antimicrob. Chemother.
28:1-12[Free Full Text].
|
| 17.
|
Munoz, R., and A. G. De La Campa.
1996.
ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of fluoroquinolones and cooperates with DNA gyrase A subunit in forming resistance phenotype.
Antimicrob. Agents Chemother.
40:2252-2257[Abstract].
|
| 18.
|
Murray, B. E.
1990.
The life and times of the Enterococcus.
Clin. Microbiol. Rev.
3:46-65[Abstract/Free Full Text].
|
| 19.
|
National Committee for Clinical Laboratory Standards.
1997.
Methods for dilution antimicrobial tests for bacteria that grow aerobically. Approved standard M7-A4.
National Committee for Laboratory Standards, Wayne, Pa.
|
| 20.
|
Ng, E. Y.,
M. Trucksis, and D. C. Hooper.
1994.
Quinolone resistance mediated by norA: physiologic characterization and relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus chromosome.
Antimicrob. Agents Chemother.
38:1345-1355[Abstract/Free Full Text].
|
| 21.
|
Pan, X. S.,
J. Ambler,
S. Mehtar, and L. M. Fisher.
1996.
Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae.
Antimicrob. Agents Chemother.
40:2321-2326[Abstract].
|
| 22.
|
Pan, X. S., and L. M. Fisher.
1996.
Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance.
J. Bacteriol.
178:4060-4069[Abstract/Free Full Text].
|
| 23.
|
Pan, X. S., and L. M. Fisher.
1998.
DNA gyrase and topoisomerase IV are dual targets of clinafloxacin action in Streptococcus pneumoniae.
Antimicrob. Agents Chemother.
42:2810-2816[Abstract/Free Full Text].
|
| 24.
|
Perichon, B.,
J. Tankovic, and P. Courvalin.
1997.
Characterization of a mutation in the parE gene that confers fluoroquinolone resistance in Streptococcus pneumoniae.
Antimicrob. Agents Chemother.
41:1166-1167[Abstract].
|
| 25.
|
Sahm, D. F., and G. T. Koburov.
1989.
In vitro activities of quinolones against enterococci resistant to penicillin-aminoglycoside synergy.
Antimicrob. Agents Chemother.
33:71-77[Abstract/Free Full Text].
|
| 26.
|
Schmitz, F. J.,
M. E. Jones,
B. Hofmann,
B. Hansen,
S. Scheuring,
M. Luckefahr,
A. Fluit,
J. Verhoef,
U. Hadding,
H. P. Heinz, and K. Kohrer.
1998.
Characterization of grlA, grlB, gyrA, and gyrB mutations in 116 unrelated isolates of Staphylococcus aureus and effects of mutations on ciprofloxacin MIC.
Antimicrob. Agents Chemother.
42:1249-1252[Abstract/Free Full Text].
|
| 27.
|
Schmitz, F. J.,
E. Lindenlauf,
B. Hofmann,
A. C. Fluit,
J. Verhoef,
H. P. Heinz, and M. E. Jones.
1998.
The prevalence of low- and high-level mupirocin resistance in staphylococci from 19 European hospitals.
J. Antimicrob. Chemother.
42:489-495[Abstract/Free Full Text].
|
| 28.
|
Tankovic, J.,
F. Mahjoubi,
P. Courvalin,
J. Duval, and R. Leclercq.
1996.
Development of fluoroquinolone resistance in Enterococcus faecalis and role of mutations in the DNA gyrase gyrA gene.
Antimicrob. Agents Chemother.
40:2558-2561[Abstract].
|
| 29.
|
Thomson, K. S., and C. C. Sanders.
1998.
The effects of increasing levels of quinolone resistance on in-vitro activity of four quinolones.
J. Antimicrob. Chemother.
42:179-187[Abstract/Free Full Text].
|
| 30.
|
Yoshida, H.,
M. Bogaki,
M. Nakamura, and S. Nakamura.
1990.
Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli.
Antimicrob. Agents Chemother.
34:1271-1272[Abstract/Free Full Text].
|
| 31.
|
Zhanel, G. G.,
J. A. Karlowsky, and D. J. Hoban.
1998.
In vitro activities of six fluoroquinolones against Canadian isolates of vancomycin-sensitive and vancomycin-resistant Enterococcus species.
Diagn. Microbiol. Infect. Dis.
31:343-347[Medline].
|
Antimicrobial Agents and Chemotherapy, October 1999, p. 2513-2516, Vol. 43, No. 10
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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