![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, December 1998, p. 3293-3295, Vol. 42, No. 12
Department of Urology, Gifu University School
of Medicine, Gifu, Japan
Received 18 June 1998/Returned for modification 13 August
1998/Accepted 9 September 1998
We examined 22 clinical isolates of Staphylococcus
epidermidis to analyze the association of alterations in GyrA and
ParC with fluoroquinolone resistance. The simultaneous presence of GyrA
and ParC alterations was associated with a high level of fluoroquinolone resistance in the clinical isolates of S. epidermidis.
Staphylococcus
epidermidis is a resident on human skin that is now recognized as
being an important opportunistic pathogen that may cause human
infection. Recently, we have observed an increase in the number of
urinary-tract-derived strains of S. epidermidis showing
decreased susceptibilities to fluoroquinolones. Most studies on the
mechanisms of quinolone resistance in bacterial species have focused on
the alterations of two target enzymes of quinolones, DNA gyrase and DNA
topoisomerase IV (2, 9). For this species, however, only
alterations in the GyrA subunit of DNA gyrase have been analyzed
(14). To extend the investigation of the association of
target alterations with quinolone resistance in S. epidermidis, we examined clinical isolates for mutations in the
regions of the gyrA and parC genes of this
species, which are analogous to the quinolone resistance-determining
region (QRDR) of the Escherichia coli gyrA gene
(16).
The type strain of S. epidermidis, ATCC 14990, was purchased
from the American Type Culture Collection. For this study, we selected
22 clinical isolates that were identified as S. epidermidis by the Vitek system with the Vitek GPI card (bioMérieux
Vitek, Inc., Hazelwood, Mo.) and which showed a variety of
susceptibilities to fluoroquinolones. They were isolated from 1993 through 1995 from Japanese patients with urinary tract infections
complicated by functional or anatomical disorders of the urinary tract.
The patients had not received antibiotic treatment when the isolates were recovered; however, sufficient information was not available regarding prior use of quinolones. We determined the susceptibilities of these isolates to norfloxacin, ofloxacin, and ciprofloxacin by the
twofold agar dilution method (6). The MICs were defined as
the lowest drug concentrations that completely inhibited visible growth
of the inoculum after incubation for 18 h at 37°C
(6).
The sequence of the parC gene of S. epidermidis
has not been reported. To determine the region of the S. epidermidis parC gene which is analogous to the QRDR of the
E. coli gyrA gene (16), we amplified a DNA
fragment from the chromosomal DNA of the type strain by PCR with two
primers named SA-PARC-A and SA-PARC-B. The primers were identical to
nucleotide positions 2171 to 2190 and 2347 to 2367, respectively, of
the Staphylococcus aureus grlA gene (3). The PCR
product was sequenced as described previously (1). In brief,
ligation of the PCR product to the vector, transformation into
competent cells, and selection of colorless recombinant colonies were
performed with the TA Cloning Kit (Invitrogen, San Diego, Calif.). Both
strands of the plasmid DNA including the PCR product were sequenced
with a nonradioactive cycle sequencing kit that included the M13
forward primer and the M13 reverse primer (Toyobo, Osaka, Japan).
To detect mutations in the gyrA gene region of S. epidermidis corresponding to the QRDR of the E. coli
gyrA gene, DNA fragments were amplified from chromosomal DNAs of
the type strain and the clinical isolates of S. epidermidis
by PCR with the primers SE-GYRA-A and SE-GYRA-B. The primer sequences
were identical to the nucleotide sequences of the S. epidermidis
gyrA gene with nucleotide positions equivalent to positions 2353 to 2372 and 2453 to 2472, respectively, of the S. aureus
gyrA gene (8). To examine the clinical isolates of
S. epidermidis for mutations in the parC gene,
DNA fragments were also amplified by PCR with primers SA-PARC-A and
SA-PARC-B. All the amplified DNA fragments were sequenced as described above.
The primers SA-PARC-A and SA-PARC-B produced a 197-bp DNA fragment from
the DNA of the S. epidermidis type strain. The 52-amino-acid sequence deduced from the nucleotide sequence of the amplified 156-bp
DNA fragment between the primers showed 96% identity with the
corresponding region of the S. aureus GrlA subunit (Fig.
1). In fluoroquinolone-resistant strains
of S. aureus, amino acid changes have been commonly observed
at Ser-80 and Glu-84 in GrlA (3, 4, 10, 15). The amino acid
in ParC of the S. epidermidis type strain equivalent to
Ser-80 of S. aureus GrlA was serine, but the amino acid
equivalent to Glu-84 of GrlA was aspartate.
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Alteration in the GyrA Subunit of DNA Gyrase and the ParC Subunit
of DNA Topoisomerase IV in Quinolone-Resistant Clinical Isolates of
Staphylococcus epidermidis
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
View larger version (29K):
[in a new window]
FIG. 1.
Comparisons of the nucleotide sequence of the amplified
DNA fragment of S. epidermidis ATCC 14990 (SepparC) with the corresponding sequence of the S. aureus grlA gene (SaugrlA) and of the deduced amino
acid sequence (SepParC) with the corresponding sequence of the S. aureus GrlA protein (SauGrlA). Primer sequences are excluded from
the comparisons. The dashes in the lines for the SaugrlA and
SauGrlA sequences indicate the nucleotides and amino acids which are
identical to the nucleotides in SepparC and the amino acids
in SepParC.
Of the 22 clinical isolates of S. epidermidis examined in
this study, the 9 isolates for which the MICs of norfloxacin,
ofloxacin, and ciprofloxacin were 
12.5, 1.56, and 1.56 µg/ml,
respectively, had no alterations in the determined regions of either
GyrA or ParC (Table 1). Conversely, all
13 isolates for which MICs of norfloxacin, ofloxacin, and ciprofloxacin
were 
12.5, 1.56, and 1.56 µg/ml, respectively, had amino acid
changes at Ser-84 and Glu-88 in GyrA and at Ser-80 and Glu-84 in ParC.
|
Of the 13 isolates with decreased susceptibilities to fluoroquinolones, one had a single amino acid change in ParC with no alteration in GyrA. The remaining 12 isolates had changes in both GyrA and ParC. The level of fluoroquinolone resistance in the single-alteration isolate was between that of isolates with alterations in neither GyrA nor ParC and that of the isolates with alterations in both GyrA and ParC. The increases in levels of fluoroquinolone resistance appeared to be associated with accumulation of amino acid changes in GyrA and ParC.
In this study, we did not examine the isolates for alterations in GyrB or ParE. We do not exclude the possibility that GyrB and/or ParE alterations could contribute to the decreased susceptibilities to fluoroquinolones of these isolates. In addition, recent studies on fluoroquinolone resistance in Streptococcus pneumoniae and S. aureus have reported that either DNA gyrase or topoisomerase IV can be the primary target of fluoroquinolones (5, 13). However, the findings obtained in the present study suggest that ParC alterations may be the primary ones associated with fluoroquinolone resistance in S. epidermidis as well as in other gram-positive bacterial species (4, 7, 10-12, 15). This study also implies that the simultaneous presence of alterations in GyrA and ParC may be responsible for the development of higher-level fluoroquinolone resistance in clinical isolates of S. epidermidis.
Nucleotide sequence accession number. The partial sequence of the S. epidermidis parC gene reported here appears in the DDBJ, EMBL, and GenBank nucleotide sequence databases under the accession no. AB015166.
| |
ACKNOWLEDGMENTS |
|---|
We thank Kyoko Hirata for technical assistance and laboratory analysis.
This study was supported in part by a Grant-in-Aid for Scientific Research (10671466) from the Ministry of Education, Science, Sports and Culture, Japan.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Urology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu City, Gifu 500-8705, Japan. Phone: 81-58-265-1241. Fax: 81-58-265-9009. E-mail: deguchit{at}cc.gifu-u.ac.jp.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
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 |
| 2. | Drlica, K., and X. Zhao. 1997. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev. 61:377-392[Abstract]. |
| 3. | Ferrero, L., B. Cameron, B. Manse, D. Lagneaux, J. Crouzet, A. Famechon, and F. Blanche. 1994. Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones. Mol. Microbiol. 13:641-653[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. |
Fournier, B., and D. C. Hooper.
1998.
Mutations in topoisomerase IV and DNA gyrase of Staphylococcus aureus: novel pleiotropic effects on quinolone and coumarin activity.
Antimicrob. Agents Chemother.
42:121-128 |
| 6. | Japan Society of Chemotherapy. 1981. A method for determining the minimum inhibitory concentrations of antimicrobial agents, 3rd ed. Chemotherapy (Tokyo) 29:76-79. (In Japanese.) |
| 7. |
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 |
| 8. |
Margerrison, E. E.,
R. Hopewell, and L. M. Fisher.
1992.
Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus encoding the DNA gyrase and B proteins.
J. Bacteriol.
174:1596-1603 |
| 9. | Nakamura, S. 1997. Mechanisms of quinolone resistance. J. Infect. Chemother. 3:128-138. |
| 10. | Ng, E. Y., M. Trucksis, and D. Hooper. 1996. Quinolone resistance mutations in topoisomerase IV: relationship to flqA locus and genetic evidence that topoisomerase IV is the primary target and DNA gyrase is the secondary target of fluoroquinolones in Staphylococcus aureus. Antimicrob. Agents Chemother. 40:1881-1888[Abstract]. |
| 11. | 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]. |
| 12. |
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 |
| 13. | Pan, X.-S., and L. M. Fisher. 1997. Targeting of DNA gyrase in Streptococcus pneumoniae by sparfloxacin: selective targeting of gyrase or topoisomerase IV by quinolones. Antimicrob. Agents Chemother. 41:471-474[Abstract]. |
| 14. |
Sreedharan, S.,
L. R. Peterson, and L. M. Fisher.
1991.
Ciprofloxacin resistance in coagulase-positive and -negative staphylococci: role of mutations at serine 84 in the DNA gyrase A protein of Staphylococcus aureus and Staphylococcus epidermidis.
Antimicrob. Agents Chemother.
35:2151-2154 |
| 15. | Yamagishi, J., T. Kojima, Y. Oyamada, K. Fujimoto, H. Hattori, S. Nakamura, and M. Inoue. 1996. Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 40:1157-1163[Abstract]. |
| 16. |
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 |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Clin. Vaccine Immunol. | Clin. Microbiol. Rev. |
|---|---|
| J. Clin. Microbiol. | ALL ASM JOURNALS |
