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Antimicrobial Agents and Chemotherapy, August 1999, p. 2051-2055, Vol. 43, No. 8
Eijkman-Winkler Institute, Utrecht
University, 3584 CX, Utrecht, The Netherlands,1
and Institute for Medical Microbiology and Virology, University
Hospital Düsseldorf, D-40225 Düsseldorf,
Germany2
Received 15 March 1999/Returned for modification 4 May
1999/Accepted 24 May 1999
The in vitro activities of ciprofloxacin, clinafloxacin,
gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were tested
against 72 ciprofloxacin-resistant and 28 ciprofloxacin-susceptible isolates of Klebsiella pneumoniae, Klebsiella
oxytoca, Enterobacter cloacae, and Enterobacter
aerogenes. Irrespective of the alterations in GyrA and ParC
proteins, clinafloxacin exhibited greater activity than all other
fluoroquinolones tested against K. pneumoniae and E. aerogenes.
Fluoroquinolones are broad-spectrum
antimicrobial agents. However, resistance to fluoroquinolones has been
increasingly reported in many bacterial species, including those of the
Enterobacteriaceae (1, 3, 12). The main
resistance mechanism in these bacteria is an alteration of the GyrA
subunit of DNA gyrase (5, 11, 20, 21). Mutations in
parC, which encodes the ParC subunit of topoisomerase IV,
seem to play a secondary role in a great variety of enterobacterial
species, including Klebsiella pneumoniae and
Enterobacter cloacae (4, 6-8, 15, 16); however,
they have not yet been investigated in Klebsiella oxytoca
and Enterobacter aerogenes.
The purpose of this study was to compare the in vitro activities of
ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin,
and trovafloxacin against isolates of Klebsiella spp. and
Enterobacter spp. with characterized gyrA and
parC genes and to analyze the prevalence of alterations in
those genes within a representative European strain collection of these species.
The isolates tested were sent from 24 European university
hospitals participating in the European SENTRY Antimicrobial
Surveillance Program, which is implemented in 13 European
countries (13, 19). Only one isolate per patient was
submitted. Between April 1997 and October 1998, 465 K. pneumoniae isolates, 148 K. oxytoca isolates, and 501 Enterobacter sp. isolates were collected. Of these,
4.7% of K. pneumoniae, 2.8% of K. oxytoca, and
10.8% of Enterobacter spp. were intermediately or fully
resistant to ciprofloxacin (MICs greater than 1 µg/ml). Most of these
ciprofloxacin-resistant isolates (22 K. pneumoniae, 4 K. oxytoca, 22 E. cloacae, and 24 E. aerogenes isolates) as well as some randomly selected
ciprofloxacin-susceptible isolates were included in the study.
Ciprofloxacin-resistant K. pneumoniae isolates were found in
six countries, including Greece (6 isolates of 39), France (1 of 83),
Italy (3 of 29), Poland (6 of 18), Spain (3 of 68), and Turkey (3 of
31). Ciprofloxacin-resistant K. oxytoca isolates were found
in Belgium (1 of 9 isolates), Greece (1 of 7), and Italy (2 of 15).
Ciprofloxacin-resistant isolates of E. cloacae were found in
Austria (1 of 7 isolates), Belgium (1 of 6), France (7 of 63),
Germany (9 of 40), Italy (2 of 27), and Portugal (2 of 21).
Ciprofloxacin-resistant E. aerogenes isolates were found in
Austria (1 of 3 isolates), Belgium (4 of 8), France (7 of 54),
Germany (4 of 6), Greece (2 of 23), Italy (3 of 14), Portugal (2 of 8),
and the United Kingdom (1 of 6). Approximately half of the
ciprofloxacin-resistant isolates came from blood cultures; the rest
were isolated from urinary tract infections, wound infections, and
nosocomial pneumonia.
MICs were determined by a broth microdilution method defined by the
National Committee for Clinical Laboratory Standards (17).
Prepared bacterial DNA was used as the template for PCR amplification
of the gyrA and parC portions homologous to the
quinolone resistance-determining region (QRDR) of Escherichia
coli (14, 21). Primers gyrA-A
(5'-CGCGTACTATACGCCATGAACGTA-3') and gyrA-C (5'-ACCGTTGATCACTTCGGTCAGG-3') were used for
Klebsiella spp. Primers parC-A
(5'-CTGAATGCCAGCGCCAAATT-3') and parC-C
(5'-TGCGGTGGAATATCGGTCGC-3') were used for K. pneumoniae. Primers parC-F1
(5'-ATGGACCGTGCGTTGCCGTTTAT-3') and parC-R1
(5'-CGGCAATACCGGTGGTGCCGTT-3') were used for K. oxytoca. For the amplification of gyrA and
parC of Enterobacter spp., we used the primers
previously defined by Deguchi et al. (8).
PCR conditions were as described previously (18), with an
annealing temperature of 55°C for Enterobacter spp. and
50°C for Klebsiella spp. PCR-amplified DNA was
sequenced by the dye terminator method in both directions using
the Ready Reaction Dye Terminator Cycle sequencing kit
(Perkin-Elmer).
Sequences with no mutations were identified on the basis of their being
identical to the published sequences of the gyrA and the
parC genes (7-9, 20). Strain NCTC49141 was
sequenced for parC as a reference strain for K. oxytoca.
The amino acid changes identified in Klebsiella spp.
and Enterobacter spp. GyrA and ParC proteins are shown
in Table
1.
Most of the ciprofloxacin-resistant K. pneumoniae isolates
demonstrated an amino acid change within the GyrA protein from Ser-83
to Tyr or Phe, as previously reported (7, 20), and one
showed a change from Ser-83 to Ile. All K. oxytoca isolates
showed a change from Thr-83 to Ile, in agreement with Weigel et al.
(20). Similarly, all resistant isolates of E. cloacae showed a change from Ser-83 to Phe or Tyr, as described
previously (8, 20), and isolates of E. aerogenes
showed a change from Ser-83 to Ile (20) or Tyr. Six
resistant K. pneumoniae isolates also showed a change from Asp-87 to Asn (7, 20) or Tyr, while one K. oxytoca isolate showed a newly described change from Asp-87 to
Gly. An Asp-87 change to Asn, Gly, or His was also found in 21 E. cloacae isolates (8), while one E. aerogenes
isolate showed a newly described change from Asp-87 to Asn. Other amino
acid changes were also observed in Ala-67, Asp-72, and Ile-78 in
E. aerogenes (Table 1).
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Comparative In Vitro Activities of Ciprofloxacin, Clinafloxacin,
Gatifloxacin, Levofloxacin, Moxifloxacin, and Trovafloxacin against
Klebsiella pneumoniae, Klebsiella oxytoca,
Enterobacter cloacae, and Enterobacter
aerogenes Clinical Isolates with Alterations in GyrA and
ParC Proteins
ABSTRACT
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TABLE 1.
Amino acid changes within GyrA and ParC and corresponding
MICs of six fluoroquinolones
With regard to the ParC protein, all ciprofloxacin-resistant Enterobacter isolates showed a change from Ser-80 to Ile or Arg. In contrast, only 10 K. pneumoniae isolates showed a change, 8 from Ser-80 to Ile and 2 from Glu-84 to Lys; the other 12 had no mutation in this gene. Only two of the four K. oxytoca isolates showed an amino acid change, from Ser-80 to Arg or Ile.
We confirmed the pattern of mutations previously described for Klebsiella spp. (7) with some isolates having only mutations in gyrA. In contrast to Japanese (8) and American (20) isolates, all European ciprofloxacin-resistant Enterobacter spp. isolates showed an alteration in ParC together with amino acid changes in GyrA. Since we sequenced the QRDR of nearly all ciprofloxacin-resistant Enterobacter sp. isolates found in this study, our results may reflect a shift toward isolates with more mutations in the present European populations of these species or a geographic difference with Japan and the United States.
Mutations or combinations of mutations found within the gyrA
or parC genes and the corresponding MICs of each of the
quinolones tested are shown in Table 1. Isolates with no identifiable
mutations within the gyrA or parC genes
demonstrated MICs of ciprofloxacin of 
0.06 µg/ml for K. pneumoniae, 0.06 to 0.25 µg/ml for K. oxytoca, 0.06 µg/ml for E. cloacae, and 0.5 to 1 µg/ml for
E. aerogenes. For the ciprofloxacin-susceptible isolates,
however, there was no clear difference in efficacy between the
different quinolones for the concentrations tested.
In Klebsiella spp., an alteration in Ser-83 of GyrA alone
was associated with a MIC of ciprofloxacin of 
2 µg/ml. Additional changes in either Asp-87 of GyrA or Ser-80 of ParC generally increased the MICs. This illustrates the major role played by alterations in
amino acid Ser-83 of GyrA and the secondary role of additional changes,
as previously reported (4, 6, 15, 16, 21).
In Enterobacter spp., combined amino acid changes in GyrA do not seem to result in higher MICs compared to single changes. Furthermore, as we observed only isolates with changes in both GyrA and ParC, the impact of changes in ParC on MICs, in addition to changes in GyrA, is difficult to define.
Since we have not examined the GyrB and ParE subunits, the possibility that some isolates have alterations in these proteins cannot be excluded.
This study is one of the few in which the in vitro activities of the newer fluoroquinolones have been simultaneously compared for Klebsiella and Enterobacter isolates with defined alterations in gyrA and parC. For all ciprofloxacin-resistant isolates tested, clinafloxacin MICs were generally two dilution steps lower than those of the second active quinolone tested. This very general observation is true for all combinations of mutations in gyrA and parC. New quinolones with improved in vitro activity should be active against both ciprofloxacin-susceptible and ciprofloxacin-resistant isolates. Based on a breakpoint of >1 µg/ml, 16 to 22 K. pneumoniae isolates were resistant to gatifloxacin, trovafloxacin, moxifloxacin, levofloxacin, and ciprofloxacin, whereas only 7 were clinafloxacin resistant. No K. oxytoca isolate was resistant to clinafloxacin, but four were resistant to the other quinolones. Six E. aerogenes isolates were clinafloxacin resistant, while 24 were resistant to the other quinolones. Finally, 20 E. cloacae isolates were resistant to clinafloxacin and 22 were resistant to the other quinolones under study. The number of isolates with MICs of <1 µg/ml was significantly greater for clinafloxacin than for the other quinolones, according to the chi-square test, for K. pneumoniae (P < 0.02) and for E. aerogenes (P < 0.001). No difference was found for E. cloacae, and due to the low number of isolates the test was not performed for K. oxytoca.
This improved in vitro activity against isolates with alterations in GyrA and ParC might be related to the interaction of the C-8 chlorine atom of this compound with the gyrase enzyme (10).
In summary, clinafloxacin shows the best in vitro activity against all species tested, independent of the genetic constitution of the gyrA and parC genes. These results echo the improved efficacy of clinafloxacin reported previously for genetically undefined isolates of these species (2). Clinafloxacin may, therefore, be of clinical value in the therapy of Klebsiella spp. and E. aerogenes infections, especially those caused by resistant strains with alterations in GyrA and ParC. However, it seems to have no improved value against E. cloacae.
Nucleotide sequence accession number. The partial sequence of the parC gene of K. oxytoca NCTC49191 was assigned EMBL accession number AJ133197.
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ACKNOWLEDGMENTS |
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We are grateful to Babak Aliyary Ghraboghly for technical help in sequencing. We thank Marita Hautvast, Mirjam Klootwijk, Karlijn Kusters, and Stefan de Vaal for their expert technical assistance.
S.B. was supported by a European Community Human Capital Mobility Grant. This work was funded in part by Bristol-Myers Squibb Pharmaceuticals via the SENTRY Antimicrobial Surveillance Program.
We thank the following members of the SENTRY participants group for referring isolates from their institutes for use in this study: Jacques Acar, Rogelio Martin Alvarez, Fernando Baquero, Jacques Bille, Dario Costa, René Courcol, Franz Daschner, Jérome Etienne, Gary French, Fred Goldstein, Deniz Gür, Ulrich Hadding, Piotr Heczko, Waleria Hryniewicz, Vincent Jarlier, Volkan Korten, Nikos Legakis, Carlo Mancini, Helmut Mittermayer, Evilio Perea, Gian-Carlo Schito, Marc Struelens, and Serhat Unal.
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FOOTNOTES |
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* Corresponding author. Mailing address: Eijkman-Winkler Institute, Utrecht University, AZU G04.614, Heidelberglaan 100, 3584 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 |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Alarcon, T.,
J. Pita,
M. Lopez-Brea, and L. J. Piddock.
1993.
High-level quinolone resistance amongst clinical isolates of Escherichia coli and Klebsiella pneumoniae from Spain.
J. Antimicrob. Chemother.
32:605-609 |
| 2. |
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 |
| 3. |
Bauernfeind, A.,
M. Abele-Horn,
P. Emmerling, and R. Jungwirth.
1994.
Multiclonal emergence of ciprofloxacin-resistant clinical isolates of Escherichia coli and Klebsiella pneumoniae.
J. Antimicrob. Chemother.
34:1074-1076 |
| 4. | Belland, R. J., S. G. Morrison, C. Ison, and W. M. Huang. 1994. Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone-resistant isolates. Mol. Microbiol. 14:371-380[Medline]. |
| 5. |
Deguchi, T.,
M. Yasuda,
M. Asano,
K. Tada,
H. Iwata,
H. Komeda,
T. Ezaki,
I. Saito, and Y. Kawada.
1995.
DNA gyrase mutations in quinolone-resistant clinical isolates of Neisseria gonorrhoeae.
Antimicrob. Agents Chemother.
39:561-563 |
| 6. | Deguchi, T., M. Yasuda, M. Nakano, S. Ozeki, T. Ezaki, I. Saito, and Y. Kawada. 1996. Quinolone-resistant Neisseria gonorrhoeae: correlation of alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV with antimicrobial susceptibility profiles. Antimicrob. Agents Chemother. 40:1020-1023[Abstract]. |
| 7. | Deguchi, T., A. Fukuoka, M. Yasuda, M. Nakano, S. Ozeki, E. Kanematsu, Y. Nishino, S. Ishihara, Y. Ban, and Y. Kawada. 1997. Alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in quinolone-resistant clinical isolates of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 41:699-701[Abstract]. |
| 8. |
Deguchi, T.,
M. Yasuda,
M. Nakano,
S. Ozeki,
E. Kanematsu,
Y. Nishino,
S. Ishihara, and Y. Kawada.
1997.
Detection of mutations in the gyrA and parC genes in quinolone-resistant clinical isolates of Enterobacter cloacae.
J. Antimicrob. Chemother.
40:543-549 |
| 9. |
Dimri, G. P., and H. K. Das.
1990.
Cloning and sequence analysis of gyrA gene of Klebsiella pneumoniae.
Nucleic Acid Res.
18:151-156 |
| 10. | Gootz, T. D., and K. E. Brighty. 1998. Chemistry and mechanism of action of the quinolone antibacterials, p. 29-80. In V. T. Andriole (ed.), The quinolones. Academic Press, San Diego, Calif. |
| 11. |
Heisig, P.,
H. Schedletzky, and H. Falkenstein-Paul.
1993.
Mutations in the gyrA gene of a highly fluoroquinolone-resistant clinical isolate of Escherichia coli.
Antimicrob. Agents Chemother.
37:696-701 |
| 12. | Hooper, D. C. 1995. Bacterial resistance to fluoroquinolones: mechanisms and patterns. Adv. Exp. Med. Biol. 390:49-57[Medline]. |
| 13. | 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]. |
| 14. | Kato, J., Y. Nishimura, R. Imamura, H. Niki, S. Hiraga, and H. Suzuki. 1990. New topoisomerase essential for chromosome segregation in E. coli. Cell 63:393-404[Medline]. |
| 15. |
Khodursky, A. B.,
E. L. Zechiedrich, and N. R. Cozzarelli.
1995.
Topoisomerase IV is a target of quinolones in Escherichia coli.
Proc. Natl. Acad. Sci. USA
92:11801-11805 |
| 16. | Kumagai, Y., J. I. Kato, K. Hoshino, T. Akasaka, K. Sato, and H. Ikeda. 1996. Quinolone-resistant mutants of Escherichia coli DNA topoisomerase IV parC gene. Antimicrob. Agents Chemother. 40:710-714[Abstract]. |
| 17. | National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial tests for bacteria that grow aerobically. Approved standard M7-A4. National Committee for Clinical Laboratory Standards, Wayne, Pa. |
| 18. |
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 |
| 19. |
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 |
| 20. |
Weigel, L. M.,
C. D. Steward, and F. C. Tenover.
1998.
gyrA mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae.
Antimicrob. Agents Chemother.
42:2661-2667 |
| 21. |
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 |
Antimicrobial Agents and Chemotherapy, August 1999, p. 2051-2055, Vol. 43, No. 8
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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