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Antimicrobial Agents and Chemotherapy, February 1998, p. 236-240, Vol. 42, No. 2
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of grlA and gyrA
Mutations in 344 Staphylococcus aureus Strains
Tong
Wang,
Mayumi
Tanaka,* and
Kenichi
Sato
New Product Research Laboratories I, Daiichi
Pharmaceutical Co., Ltd., Edogawa-ku, Tokyo 134, Japan
Received 23 June 1997/Returned for modification 15 October
1997/Accepted 25 November 1997
 |
ABSTRACT |
Mutations in the grlA and gyrA genes of 344 clinical strains of Staphylococcus aureus isolated in 1994 in Japan were identified by combinations of single-strand conformation
polymorphism analysis, restriction fragment length analysis, and direct
sequencing to identify possible relationships to fluoroquinolone
resistance. Five types of single-point mutations and four types of
double mutations were observed in the grlA genes of 204 strains (59.3%). Four types of single-point mutations and four types
of double mutations were found in the gyrA genes of 188 strains (54.7%). Among them, the grlA mutation of
TCC
TTC or TAC (Ser-80Phe or Tyr) and the gyrA
mutation of TCATTA (Ser-84Leu) were principal, being detected in
137 (39.8%) and 121 (35.9%) isolates, respectively. The
grlA point mutations of CATCAC (His-77 [silent]),
TCACCA (Ser-81Pro), and ATAATT (Ile-100 [silent]) were
novel, as was the GACGGC (Asp-73Gly) change in gyrA.
A total of 15 types of mutation combinations within both genes were
related to ciprofloxacin resistance (MIC 
3.13 µg/ml) and were
present in 193 mutants (56.1%). Strains containing mutations in both
genes were highly resistant to ciprofloxacin (MIC at which 50% of the
isolates are inhibited [MIC50] = 50 µg/ml). Those with
the Ser-80Phe or Tyr alteration in grlA but wild-type
gyrA showed a lower level of ciprofloxacin resistance
(MIC50 
12.5 µg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlA
in the presence or absence of a concomitant mutation at codon 73, 84, or 88 in gyrA (MIC 6.25 µg/ml). The new
fluoroquinolone DU-6859a showed good activity with 186 of 193 isolates
(96.4%) for which the MIC was 6.25 µg/ml.
| |
INTRODUCTION |
Staphylococcus aureus
infections, particularly those caused by methicillin-resistant S. aureus, pose serious therapeutic difficulties, because few
antimicrobial agents are effective against this pathogen. Fluoroquinolones, broad-spectrum and potent antimicrobial agents, have
been effective in the treatment of these infections. With the
increasing use of fluoroquinolones, resistance in S. aureus, especially methicillin-resistant S. aureus has become
widespread in recent years (1, 12).
Three mechanisms involved in fluoroquinolone resistance have been
proposed. One is topoisomerase IV gene mutations (4, 18), a
second is DNA gyrase gene mutations (13, 16), and a third is
an active efflux pump (8, 19). Recent studies have
demonstrated that the primary target of fluoroquinolones in S. aureus is DNA topoisomerase IV, which is composed of the GrlA and
GrlB subunits, encoded by the grlA and grlB
genes, respectively. DNA gyrase is considered a secondary target
(3, 4, 10, 15). In S. aureus clinical strains,
mutations in either the grlA or gyrA gene lead to
quinolone resistance. grlA mutations are associated with
both high- and low-level resistances, while gyrA mutations
are responsible for increases in ciprofloxacin (CPFX) resistance in
grlA mutants. A combination of mutations in both genes can
cause high-level quinolone resistance (4, 5, 13, 15, 16,
18).
Several methods have been used for detection of point mutations of
genes. Single-strand conformation polymorphism (SSCP) analysis is a
rapid, simple, and effective method in which a mutated sequence is
detected by a change in mobility during polyacrylamide gel electrophoresis caused by its altered folded structure (6). It has been applied in detection of DNA gyrase gene mutations in
S. aureus (14, 17) and Escherichia
coli (11). In SSCP analysis in our study, proper
conditions and a new system were used for detection of grlA
and gyrA mutations. By using combinations of SSCP analysis,
restriction fragment length polymorphism (RFLP) analysis, and direct
sequencing, we examined grlA and gyrA mutations in 344 S. aureus strains and studied the relationship
between combinations of mutations of both genes and susceptibility of the various mutants to three fluoroquinolones.
| |
MATERIALS AND METHODS |
Antimicrobial agents and bacterial strains.
CPFX,
levofloxacin (LVFX), and DU-6859a were synthesized at the New Product
Research Laboratories I, Daiichi Pharmaceutical Co., Ltd., Tokyo,
Japan. A total of 344 clinical S. aureus strains (one strain
per patient) were collected by LVFX surveillance groups from 24 hospitals all over Japan. These were isolated from June to November
1994 and included 215 methicillin-resistant (32 CPFX-susceptible and
183 CPFX-resistant) and 129 methicillin-susceptible (122 CPFX-susceptible and 7 CPFX-resistant) strains. S. aureus
FDA 209-P, 891185 (Ser-80Phe), and 900165 (Glu-84Lys) were used
as controls for detection of grlA mutations, while FDA
209-P, 900165 (Ser-84Leu), 6859a-r (Glu-88Lys), 87-53 (Ile-86
[silent]), and 87-20 (Ser-84Leu, Ile-86 [silent]) were used for
detection of gyrA mutations (15, 16).
Determination of MICs.
The MICs were determined by standard
agar dilution methods (9) with Mueller-Hinton agar (Difco,
Detroit, Mich.). Drug-containing agar plates were incubated with one
loopful (5 µl) of an inoculum corresponding to about 104
CFU per spot and were incubated at 37°C for 18 h. The MIC was defined as the lowest drug concentration which prevented visible growth
of bacteria.
PCR experiments.
Chromosomal DNA was prepared from 2 µl of
an overnight culture heated at 98°C for 5 min. PCR was performed with
cycling at 94°C for 30 s, 52°C for 30 s, and 70°C for 1 min for 30 cycles by using 2.5 U of recombinant Taq DNA
polymerase (Takara, Shiga, Japan). Oligonucleotides
5'-TTCCGTAAAAGTGCGAAAACAG (nucleotides 178 to 199) and
5'-CGCATTGCCGCTGGCGGATCCTTATCGATAC (complementary to
nucleotides 323 to 353) were used for amplification of a 176-bp grlA fragment. For gyrA gene amplification,
5'-CATATAAAAAATCAGCACGTATCGTT (nucleotides 188 to 213) was
used as the sense primer, and 5'-TGAGCCATACGTACCATTGC (complementary to nucleotides 265 to 284) (97-bp fragment) and 5'-CGCCATCTCCATCCATTGAACCAAA (complementary to nucleotides
328 to 352) (165-bp fragment) were used as antisense primers for SSCP analysis and direct sequencing, respectively. The amplified products were checked by agarose gel electrophoresis.
SSCP analysis.
PCR products were mixed with denaturant
solution (95% formamide, 20 mM EDTA [pH 8.0], 0.05% bromophenol
blue, 0.05% xylene cyanol). The mixtures were heated at 80°C for 5 min and then were cooled on ice immediately. Samples were then
separated by 12.5% polyacrylamide gel (Daiichi Pure Chemicals, Tokyo,
Japan) electrophoresis at 17°C, and DNA bands were visualized by
using a silver stain kit (Daiichi Pure Chemicals).
RFLP analysis.
For detection of mutations in the
grlA gene, we used restriction endonuclease HinfI
(Toyobo, Tokyo, Japan) for the Ser-80Tyr alteration and
BsrGI (New England Biolabs, Beverly, Mass.) for the
Glu-84Lys alteration. The reaction mixtures (1 U of enzyme added to
10 µl of each PCR product) were incubated at 37°C for more than
1 h, and the sizes of the resulting fragments were ascertained by
agarose gel electrophoresis.
DNA sequencing.
The PCR-amplified DNA from grlA
was cloned into the vector pGEM-T (Promega, Madison, Wis.) and
sequenced with an ALFred DNA sequencer (Pharmacia). The amplified
fragment from gyrA was sequenced directly with an AutoLoad
solid-phase sequencing kit (Pharmacia).
| |
RESULTS |
Detection of mutations in the grlA gene.
By using
a combination of SSCP, RFLP analysis, and sequencing, five single-point
mutations and four double mutations were observed in the
grlA gene in 204 S. aureus strains (59.3%)
(Table 1). Among them, the TCCTTC or
TAC (Ser-80Phe or Tyr) single-point mutation was principal; it was
detected in 137 isolates (39.8%). The double mutation of TCCTTC or
TAC and GAAAAA (Ser-80Phe or Tyr and Glu-84Lys) was found in
49 mutants (14.2%). The CATCAC (His-77) and ATAATT (Ile-100)
single-point mutations were silent mutations, i.e., they led to no
amino acid alteration. The TCACCA (Ser-81Pro) was a novel point
mutation in the grlA gene. Double-codon alterations of
Ser-80Tyr and Glu-84Lys, Ser-80Phe and Ser-81Pro, and
Ser-81Pro and Glu-84Lys were also novel grlA mutations
and were identified in 41, 1, and 1 isolates, respectively. As shown in
Fig. 1A, six of nine types of
grlA mutations were distinguishable from the wild type by
SSCP analysis.
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FIG. 1.
Detection of mutations in the grlA gene (A)
and the gyrA gene (B) by SSCP analysis. Mutations in the
genes cause amino acid changes as follows. (A) Lane 1, His-77
(CATCAC [silent]); lane 2, Ser-80Phe; lane 3, Ile-100
(ATAATT [silent]); lane 4, Ser-80Phe and Ser-81Pro; lane 5, Ser-80Phe and Glu-84Lys; lane 6, Ser-81Pro and Glu-84Lys;
lane 7, none (wild type). (B) Lane 1, none (wild type); lane 2, Ser-84Leu; lane 3, Ser-84Leu and Ile-86 (ATTATC [silent]);
lane 4, Ile-86 (silent); lane 5, Ser-84Leu and Ser-85Pro; lane 6, Asp-73Gly; lane 7, Asp-73Gly and Ser-84Leu; lane 8, Ser-84Leu and Glu-88Lys; lane 9, Glu-88Lys. Arrowheads
indicate the bands used to distinguish differences.
|
|
Detection of mutations in the gyrA gene.
As shown
in Table 2, 188 S. aureus
strains (54.7%) contained mutations in the gyrA gene. By
SSCP analysis and direct sequencing, four types of single-point
mutations and four types of double mutations were detected at codons
73, 84, 86 (silent), 88, 84 and 73, 84 and 85, 84 and 86, and 84 and
88. The single-point mutations of TCATTA (Ser-84Leu) and
GAAAAA (Glu-88Lys) were principal; they were found in 121 (35.0%) and 36 (10.5%) isolates, respectively. The GACGGC
(Asp-73Gly) change was a novel point mutation in the
gyrA gene. The eight types of gyrA mutants and the wild type had distinct SSCP patterns (Fig. 1B); however, the SSCP
patterns in lanes 6 (alteration at codon 73) and 7 (alterations at
codons 73 and 84) of Fig. 1B were similar, so the mutations of four
strains were confirmed by sequencing. There was no strain which
possessed amino acid changes in GyrA in the absence of a grlA mutation.
Susceptibilities of mutants to quinolones.
A total of 19 mutation combinations were found in the grlA and
gyrA genes of the 344 S. aureus strains (Table
3). Isolates with grlA-gyrA
mutation combinations of codon 77-none, codon 100-none, and
none-codon 86 were found to have quinolone susceptibilities similar to
that of the wild type, because these silent mutations are not
responsible for quinolone resistance. The other 15 types observed in
193 mutants (56.1%) were related to CPFX resistance (MIC 3.13 µg/ml). Overall, those strains mutated in both genes showed
higher-level CPFX resistance. The MIC at which 50% of the isolates are
inhibited (MIC50) and MIC90 were 50 and 800 µg/ml, respectively. Among such strains, those with combined
mutations at codons 84-84, 84-84 plus 86, 80 plus 84-84, 80 plus
84-73 plus 84, 80 plus 84-84 plus 86, 80 plus 84-84 plus 85, and 81 plus 84-84 were highly resistant to CPFX (MIC 100 µg/ml).
The combination of the Ser-80Phe or Tyr alteration in
grlA but wild-type gyrA was present in strains
with lower-level resistance to CPFX. The MIC range, MIC50,
and MIC90 for these strains were 3.13 to 12.5, 12.5, and
12.5 µg/ml, respectively.
With respect to LVFX and DU-6859a, the distribution of susceptibilities
of mutants had a tendency similar to that of susceptibility
to CPFX
(Fig.
2). LVFX was active against 68 of 193 mutants (35.2%)
which contained alterations codon as codon
80-73, 80-84, 80-88,
or 80-none (MIC of LVFX
6.25 µg/ml).
The other mutants were
moderately or highly resistant to LVFX. DU-6859a
had good activity
against 120 of the mutants (62.2%; MIC of
DU-6859a
0.78 µg/ml).
The MICs of DU-6859a for 66 of the
mutants (34.2%) were from 1.56
to 6.25 µg/ml. Six mutants with the
codon 80 plus 84-84 plus 85
alterations and one with the codon 84-84
plus 88 alterations were
highly resistant to DU-6859a (MIC
25 µg/ml).
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|
FIG. 2.
Distribution of the MICs of LVFX and DU-6859a versus the
MICs of ciprofloxacin. The sizes of the circles reflect the numbers of
strains.
|
|
| |
DISCUSSION |
In clinical S. aureus strains, five types of
grlA mutations (causing Ser-80Phe, Ser-80Tyr,
Glu-84Lys, Glu-84Gln, and Ser-80Phe plus Glu-84Lys
changes) were demonstrated to be responsible for quinolone resistance
(2-4, 10, 15, 18). In gyrA, 10 types of
mutations (causing Ser-84Leu, Ser-84Ala, Ser-84Val,
Ser-85Pro, Glu-88Lys, Glu-88Gly, Ser-84Leu plus
Ser-85Pro, Ser-84Leu plus Ile-86 [silent], Ser-84Leu plus
Glu-88Lys, and Ser-84Leu plus Glu-88Gly changes) were
found in strains with high-level quinolone resistance (5, 7,
13-17).
In this study, we examined 344 clinical isolates of S. aureus by a combination of SSCP, RFLP analysis, and direct
sequencing and found nine types of mutations in grlA and
eight types in gyrA. Among them, five types in
grlA and two types in gyrA were novel. The
grlA mutation at codon 80 and the gyrA mutation
at codon 84 were principal, being found in 137 of 204 GrlA mutants
(67.2%) and 121 of 188 GyrA mutants (64.4%), respectively. This
result is consistent with findings reported for clinical isolates
(4, 14). grlA mutations at codons 80 and 84 and
gyrA mutations at codons 84, 85, and 88 were detected in the
CPFX-resistant strains (MIC 3.13 µg/ml), which is in
agreement with other reports that these point mutations are responsible
for quinolone resistance (5, 18). Further study is necessary
to determine whether the Ser-81Pro alteration encoded by
grlA and the Asp-73Gly alteration encoded by
gyrA directly contributed to quinolone resistance.
Fifteen types of mutation combinations of both genes observed in this
investigation were related to CPFX resistance. Among them, 13 mutants
with lower-level CPFX resistance contained a grlA mutation
at codon 80 but no mutation in gyrA. This supports the
notion that in S. aureus topoisomerase IV is a primary
target of fluoroquinolones. The finding that grlA-gyrA
double mutants exhibited higher-level CPFX resistance
(MIC50 = 50 µg/ml) confirmed previous genetic studies
(10, 18). The combinations of mutations including the
Glu-84Lys alteration encoded by grlA and the Ser-84Leu alteration encoded by gyrA conferred high-level quinolone
resistance (MIC of CPFX 100 µg/ml).
Diversity in the MICs of fluoroquinolones for strains with same
mutation combination suggests that the strains may possess other
mechanisms of resistance. Some strains with no mutations in both genes
or with a silent mutation in either gene showed low-level CPFX
resistance (MIC, 1.56 to 3.13 µg/ml), which suggests that other
resistance mechanisms are present. In this study, the alteration at
codon 116 in the sequence encoding GrlA, which considered a DNA binding
site (10), was not checked because the length of the DNA
fragment was limited for detection by SSCP analysis. The amino acid
change in the remaining region of GrlA and GyrA, alteration in GrlB and
GyrB, and the association of the quinolone efflux system were possibly
the cause of the diversity in the MICs. The numerous and complicated
mutations seen may explain the rapid and widespread development of
quinolone resistance described for S. aureus. DU-6859a
showed good activity against CPFX- or LVFX-resistant mutants because of
its high inhibitory activity against both topoisomerase IV and DNA
gyrase as demonstrated by Tanaka et al. (15).
In this study, SSCP analysis was found to be a rapid, simple, and
effective method for detection of point mutations in both the
grlA gene and the gyrA gene of S. aureus strains. The phenomenon that one strand could be
separated into two bands due to different stable conformations
(6) was observed as well under our SSCP conditions.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from the Japan Health Sciences
Foundation.
We thank Yoshikuni Onodera and Takaaki Akasaka for their technical
assistance and Yuki Nagano for synthesizing the primers used in this
study.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: New
Product Research Laboratories I, Daiichi Pharmaceutical Co., Ltd.,
16-13 Kitakasai 1-Chome, Edogawa-ku, Tokyo 134, Japan. Phone:
81-3-3680-0151, ext. 5810. Fax: 81-3-5695-8344. E-mail:
KYS04512{at}niftyserve.or.jp.
| |
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Antimicrobial Agents and Chemotherapy, February 1998, p. 236-240, Vol. 42, No. 2
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
