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Antimicrobial Agents and Chemotherapy, May 1999, p. 1156-1162, Vol. 43, No. 5
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Sequence Analysis of the gyrA and
parC Homologues of a Wild-Type Strain of Vibrio
parahaemolyticus and Its Fluoroquinolone-Resistant
Mutants
Jun
Okuda,1,2
Eriko
Hayakawa,2
Mitsuaki
Nishibuchi,3 and
Takeshi
Nishino2,*
New Product Research Laboratories I, Daiichi
Pharmaceutical Co., Ltd., Edogawa-ku, Tokyo,1
and Department of Microbiology, Kyoto Pharmaceutical
University, Yamashina-ku,2 and Center
for Southeast Asian Studies, Kyoto University, Yoshida,
Sakyo-ku,3 Kyoto, Japan
Received 26 October 1998/Returned for modification 29 January
1999/Accepted 7 March 1999
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ABSTRACT |
Vibrio parahaemolyticus causes seafood-borne
gastroenteritis in humans. It is particularly important in Japan, where
raw seafood is frequently consumed. Fluoroquinolone is one of the
current drugs of choice for treating patients infected by V. parahaemolyticus because resistant strains are rarely found. To
study a possible fluoroquinolone resistance mechanism in this organism,
nucleotide sequences that are homologous to known gyrA and
parC genes have been cloned from V. parahaemolyticus AQ3815 and sequenced by amplification with
degenerate primers of the quinolone resistance-determining region
(QRDR), followed by cassette ligation-mediated PCR. Open reading frames
encoding polypeptides of 878 and 761 amino acid residues were detected
in the gyrA and parC homologues, respectively. The V. parahaemolyticus GyrA and ParC sequences were most
closely related to Erwinia carotovora GyrA (76% identity)
and Escherichia coli ParC (69% identity) sequences,
respectively. Ciprofloxacin-resistant mutants of AQ3815 were obtained
on an agar medium by multistep selection with increasing levels of the
quinolone. One point mutation only in the gyrA QRDR was
detected among mutants with low- to intermediate-level resistance,
while point mutations in both the gyrA and parC
QRDRs were detected only in strains with high-level resistance. These
results strongly suggest that, as in other gram-negative bacteria, GyrA
and ParC are the primary and secondary targets, respectively, of
ciprofloxacin in V. parahaemolyticus.
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INTRODUCTION |
Bacterial species that have
developed clinical resistance to quinolones are increasing in numbers,
and the mechanisms of their resistance have been studied. Most of the
acquired resistance can be attributed to mutations in the genes
encoding DNA gyrase or topoisomerase IV (Topo IV) (30).
DNA gyrase, a type II DNA topoisomerase, catalyzes ATP-dependent
negative supercoiling of DNA and is involved in DNA replication, recombination, and transcription (43). The enzyme consists
of GyrA and GyrB subunits, encoded by the gyrA and
gyrB genes, respectively (1, 39, 46). The two
genes are unlinked in Escherichia coli (39, 46)
and map to 48 (gyrA) and 83 (gyrB) min on the chromosomal map (3) but are contiguous in
Staphylococcus aureus (16). GyrA is considered to
be responsible for DNA strand cleavage and rejoining (14,
43), and GyrB contains ATPase activity (41). Topo IV
is a type II topoisomerase composed of ParC and ParE subunits, the
amino acid sequences of which are homologous to some degree with those
of GyrA and GyrB, respectively (20, 35). Topo IV has DNA
decatenating and relaxing activities and plays an essential role in
partitioning chromosomes at the terminal stage of chromosome
replication (2). The majority of the quinolone resistance
mutations in 17 bacterial species have been shown to map to a
relatively small region at the N terminus of GyrA, corresponding to the
67th through the 106th amino acid residues in E. coli K-12; this region is called the quinolone resistance-determining region (QRDR) (30, 47). Quinolone-resistance mutations in the
parC genes of E. coli, Klebsiella
pneumoniae, Neisseria gonorrhoeae, S. aureus, and Streptococcus pneumoniae have been
analyzed; the mutations were detected in the region corresponding to
the QRDR of GyrA (30).
High-level resistance to quinolones appears to emerge in a stepwise
fashion. E. coli and N. gonorrhoeae strains with
low-level resistance to quinolones have a gyrA mutation
alone, and strains with high-level resistance possess both
gyrA and parC mutations (5, 15).
Therefore, GyrA and ParC are considered to be the primary and secondary
targets, respectively, of quinolones in these organisms. On the other
hand, ParC (GrlA) and GyrA seem to be the primary and secondary
targets, respectively, of ciprofloxacin in S. aureus
(9, 10, 30).
Vibrio parahaemolyticus is a gram-negative marine bacterium.
Strains producing a thermostable direct hemolysin or related hemolysins
can cause gastroenteritis in humans who eat contaminated seafood
(31, 32). This is of particular importance in Japan, where
raw seafood is frequently consumed. V. parahaemolyticus is
usually susceptible to tetracycline, chloramphenicol, gentamicin, and
nalidixic acid but resistant to ampicillin, carbenicillin, and
cephalothin (18, 19, 25, 28). New quinolones such as
norfloxacin and enoxacin have potent inhibitory activity against V. parahaemolyticus (7, 29, 38).
Quinolone-resistant strains have rarely been found among strains of
V. parahaemolyticus isolated from the environment and
clinical sources. Fluoroquinolone is one of the current drugs of choice
for treating patients infected by this organism, because it is
considered that this marine bacterium is not exposed to quinolones in
its natural habitat. However, quinolone-resistant V. parahaemolyticus strains might emerge in the future if spontaneous
mutation of the gyrA or parC gene, followed by
selection of the mutant in the presence of quinolones, is the general
mechanism of quinolone resistance. To examine this hypothesis, we first
identified the gyrA and parC homologues in
V. parahaemolyticus by cassette ligation-mediated PCR, in
which well-conserved amino acid sequences of the QRDR of GyrA and the
corresponding ParC region (hereinafter called the QRDR of ParC) are
utilized. We then obtained quinolone-resistant mutants in vitro and
examined whether the mutation can be detected in the gyrA or
parC gene.
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MATERIALS AND METHODS |
Bacterial strains and growth medium.
A clinical strain of
V. parahaemolyticus, AQ3815, and its ciprofloxacin
(CIP)-resistant mutants (described below) were grown at 37°C with
Luria-Bertani (LB) broth or agar (26) unless otherwise specified. AQ3815 was isolated at Osaka Quarantine Station, Osaka, Japan, in 1983 from a traveler with diarrhea arriving from the Philippines. This strain carried the tdh genes, encoding
thermostable direct hemolysin, a major virulence factor of V. parahaemolyticus (31).
Nucleotide sequence determination of the gyrA and
parC genes.
The nucleotide sequences of the
gyrA and parC genes of V. parahaemolyticus AQ3815 were determined in three steps, as shown schematically in Fig. 1. The sequences of
the QRDRs of the genes were determined first, and then their flanking
sequences were determined by a walking method. Finally, the entire gene
sequences were determined. In each step, the target sequence was first
amplified by a PCR method, as described below. PCR products were then
cloned into vector pGEM-T (Promega, Madison, Wis.) and sequenced with BcaBEST dideoxy sequencing kit (Takara) and [
-32P]dCTP
or with the ABI-Prism Big Dye determinator cycle sequencing ready
reaction kit and an ABI-Prism 377 sequencer (Perkin-Elmer, Applied
Biosystems Division, Foster City, Calif.). The nucleotide sequences of
both strands of the cloned sequence were determined. The sequences of
the PCR primers are listed in Table 1.
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FIG. 1.
PCR strategy used to determine the nucleotide sequences
of the gyrA and parC genes. (A) gyrA
gene (boxed) and flanking sequences (straight lines). (B)
parC gene (boxed) and flanking sequences (straight lines).
The distances between the primers are not drawn to scale. Arrows
indicate locations and directions of PCR primers.
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|
To determine the QRDR sequence of the gyrA gene, the
sequence was amplified by a semi-nested PCR method (Fig. 1A, step 1). The nucleotide sequences of the degenerate primers used in PCR were
deduced from the amino acid sequences of highly conserved regions of
GyrA of Pseudomonas aeruginosa, S. aureus,
Aeromonas salmonicida, E. coli, and
Campylobacter jejuni (22, 24, 33, 39, 44). The
first PCR was carried out in a 50-µl reaction mix containing 200 µM
(each) deoxynucleoside triphosphates, 10 ng of total DNA of AQ3815, 2 µl of each primer (VGYR-1 and -4), 5 µl of GeneTaq
buffer (Wako, Osaka, Japan), and 3 U of GeneTaq polymerase
(Wako). Thermocycling was one cycle of 95°C for 5 min, followed by
three cycles of 95°C for 30 s, 37°C for 30 s, and 72°C
for 2 min, and then 30 cycles of 95°C for 30 s, 45°C for 30 s, and 72°C for 2 min. The second PCR was carried out in a 50-µl reaction mix containing 200 µM (each) deoxynucleoside
triphosphates, 2 µl of the first PCR product, 2 µl of each primer
(VGYR-2 and -4), 5 µl of GeneTaq buffer, and 3 U of
GeneTaq polymerase. Thermocycling was one cycle of 95°C
for 5 min, followed by three cycles of 95°C for 30 s, 37°C for
30 s, and 72°C for 2 min, and then 30 cycles of 95°C for
30 s, 55°C for 30 s, and 72°C for 2 min. A DNA fragment of ca. 0.2 kb was produced. This fragment was cloned and the nucleotide sequence was determined.
The upstream and downstream sequences of the
gyrA QRDR were
amplified by cassette ligation-mediated PCR (step 2 of Fig.
1A).
This
PCR method allows amplification of nucleotide sequences from
the DNA
region for which the nucleotide sequence is known (
17).
AQ3815 total DNA was digested separately with
SalI,
PstI,
HindIII,
XbaI, and
EcoRI. Each enzyme digest was ligated with the
double-stranded
ligation cassette containing the respective sticky end
for ligation
and the nucleotide sequence complementary to two PCR
primers (cassette
C1 and cassette C2). The ligation products were used
as templates
for PCR. One PCR primer pair was cassette C1 or cassette
C2, the
5' end of which was not phosphorylated. The ligation cassettes,
cassette C1, and cassette C2 were included in an LA PCR in vitro
cloning kit purchased from Takara, Shiga, Japan. The other PCR
primer
was designed so that it was complementary to a part of
the
gyrA QRDR sequence. Specific amplification is expected to
result between the cassette primer and the
gyrA
QRDR-complementary
primer because of the nonphosphorylation of the 5'
end of the
cassette primer (
17). Cassette ligation-mediated
PCR was carried
out in a nested fashion (Fig.
1A, step 2). The first
PCR was performed
in a 50-µl reaction mix containing 400 µM (each)
deoxynucleoside
triphosphates, 500 ng of template DNA (each of the
ligation products),
0.2 µM (each) primers (cassette C1 and GYRAS1 for
the QRDR upstream,
cassette C1 and GYRS1 for the QRDR downstream), 5 µl of Takara
LA PCR buffer II (Mg
2+) (Takara), and 2.5 U
of Takara LA
Taq polymerase (Takara). Takara
LA
Taq polymerase was employed to achieve long-range and
high-proofreading
PCR. Thermocycling was 30 cycles of 94°C for
30 s, 55°C for 2
min, and 72°C for 1 min. The second PCR was
performed in a 50-µl
reaction mix containing 400 µM (each)
deoxynucleoside triphosphates,
2 µl of the first PCR product, 0.2 µM (each) primers (cassette
C2 and GYRAS2 for the QRDR upstream,
cassette C2 and GYRS2 for
the QRDR downstream), 5 µl of Takara LA PCR
buffer II (Mg
2+), and 2.5 U of Takara LA
Taq
polymerase. Thermocycling was 30
cycles of 94°C for 30 s, 63°C
for 2 min, and 72°C for 1 min. Each
restriction enzyme digest of
AQ3815 DNA yielded a PCR product
of a certain size.
PstI
digest resulted in a 2-kb PCR product
for the
gyrA QRDR
upstream sequence.
HindIII digest resulted in
a 2.4-kb
PCR product for the
gyrA QRDR downstream sequence. These
PCR
products were judged to be of reasonable sizes and thus were
cloned and
the nucleotide sequences were
determined.
The determined upstream and downstream sequences of the
gyrA
gene allowed design of the PCR primers, GYRCL1 and GYRCL2, for
amplification of the sequence containing the complete
gyrA
gene
(Fig.
1A, step 3). PCR was carried out in a 50-µl reaction mix
containing 400 µM (each) deoxynucleoside triphosphates, 50 ng
of
AQ3815 total DNA, 0.2 µM (each) primers, 5 µl of Takara LA
PCR
buffer II (Mg
2+), and 2.5 U of Takara LA
Taq
polymerase. Thermocycling was 30
cycles of 94°C for 30 s, 55°C
for 2 min, and 72°C for 1 min. An
amplicon of 3 kb was cloned and the
nucleotide sequence was
determined.
The nucleotide sequence of the
parC gene was determined by
the same method as for the
gyrA sequence determination, with
minor
modifications (Fig.
1B). The modifications were as follows. To
determine the QRDR sequence of the
parC gene, the nucleotide
sequences
of the degenerate primers used in PCR were deduced from the
amino
acid sequences of highly conserved regions of ParC of
E. coli,
S. aureus, and
N. gonorrhoeae (
5,
11,
20). To amplify the
QRDR sequence, PCR primer VGYR-3 was used
in place of VGYR-2 in
the second PCR (Fig.
1B, step 1). This resulted
in an amplicon
of ca. 0.15 kb. To amplify the upstream and downstream
sequences
of the QRDR, PCR primers PARAS1, PARAS2, PARS1, and PARS2
were
used in place of GYRAS1, GYRAS2, GYRS1, and GYRS2, respectively
(Fig.
1B, step 2). An annealing temperature of 58°C was employed
in
place of 63°C in the second of the nested PCRs. As a result,
PstI digest of AQ3815 DNA yielded a 1.4-kb PCR product for
the
parC QRDR upstream sequence and
SalI digest
yielded a 2.1-kb PCR
product for the
parC QRDR downstream
sequence. These fragments
were judged to be of reasonable sizes and
thus were cloned, and
the nucleotide sequences were determined. To
amplify the sequence
containing the complete
parC gene, PCR
primers PARCL1 and PARCL2
were used in place of GYRCL1 and GYRCL2 (Fig.
1B, step 3). This
resulted in an amplicon of 2.5 kb, and this amplicon
was cloned
and the nucleotide sequence was
determined.
To compare the QRDRs of AQ3815 and its CIP-resistant mutants, the QRDR
sequences of the
gyrA and
parC genes of these
strains
were amplified as follows. PCR was carried out in a 50-µl
reaction
mix containing 200 µM (each) deoxynucleoside triphosphates,
30
µl of 10-fold-diluted boiled overnight culture, 0.2 µM (each)
primers (GYRM1 and GYRM2 for the
gyrA QRDR, PARM1 and PARM2
for
the
parC QRDR), 5 µl of Gene
Taq buffer, and
2.5 U of Gene
Taq polymerase.
Thermocycling was 30 cycles of
95°C for 30 s, 60°C for 30 s, and
72°C for 2 min. PCR
was expected to yield amplicons of 200 and
214 bp for the
gyrA QRDR and the
parC QRDR, respectively. These
amplicons were cloned and the nucleotide sequences were
determined.
MIC.
MICs were determined by the twofold agar dilution
method recommended by the Japan Society of Chemotherapy. The test
strain was grown in LB broth to mid-logarithmic phase at 37°C. The
concentration of the organism was then adjusted to 106
CFU/ml, and 5 µl of the culture was inoculated onto LB agar. The MIC
was determined after an 18-h incubation at 37°C.
Antibiotics.
The following antibiotics were purchased from
the indicated manufacturers: CIP, Bayer (Osaka, Japan); enoxacin,
Dainippon Pharmaceutical Co. (Osaka, Japan); nadifloxacin, Otsuka
Pharmaceutical Co. (Tokyo, Japan); nalidixic acid, Dainippon
Pharmaceutical Co.; norfloxacin, Kyorin Pharmaceutical Co. (Tokyo,
Japan); ofloxacin, Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan); and
sparfloxacin, Dainippon Pharmaceutical Co.
In vitro selection of CIP-resistant mutants.
CIP-resistant
mutants of AQ3815 were obtained by plating the test strain onto LB agar
containing CIP. A mutant resistant to CIP at 0.78 µg/ml was selected
first, and mutants resistant to higher concentrations of CIP (3.13, 25, and 50 µg/ml) were obtained from the mutant step by step (explained below).
Nucleotide sequence accession numbers.
The nucleotide
sequences of the gyrA and parC genes will appear
in the GenBank nucleotide sequence database under accession no.
AB023569 and AB023570, respectively.
| |
RESULTS |
Nucleotide sequences of the gyrA and parC
genes.
The nucleotide sequences of the gyrA and
parC genes of V. parahaemolyticus AQ3815 were
determined by PCR amplification, cloning, and determination of their
sequences in three steps, as illustrated in Fig. 1 and described in
Materials and Methods.
A 203-bp PCR product, for the
gyrA QRDR, and a 146-bp PCR
product, for the
parC QRDR, were amplified (Fig.
1, steps
1). To
confirm that the two fragments resulted from amplification of
the
gyrA gene and the
parC gene, respectively,
the amplified fragments
were sequenced and the deduced amino acid
sequences were compared
with GyrA and ParC of other bacterial species
so far reported.
The deduced amino acid sequence of the 203-bp fragment
had high
identity with the corresponding sequences of GyrA of
E. coli (100%),
Haemophilus influenzae (100%),
K. pneumoniae (97.5%), and
Erwinia carotovora (97.5%)
(
9,
12,
36,
39). The deduced amino
acid sequence of the
146-bp fragment showed high identity with
the corresponding sequences
of ParC of
E. coli (100%),
Salmonella typhimurium (100%),
H. influenzae (97.5%), and
N. gonorrhoeae (75%) (
5,
12,
20,
23).
The upstream and downstream sequences of the QRDR were determined in
the second step, and a stretch of the sequence containing
the complete
gene was amplified and sequenced in the final step.
There were no
discrepancies among the sequences determined in
the three steps. The
gyrA gene had a predicted open reading frame
(ORF) encoding
878 amino acids, preceded by putative promoter
and ribosome binding
sequences (data not shown). The amino acid
sequence encoded by this ORF
was homologous with that of
E. coli GyrA not only in the
QRDR but also in the other regions (Fig.
2). The amino acid
sequence identities of this ORF with GyrA and
ParC proteins of other
bacterial species were compared. So far,
complete sequences of both
GyrA and ParC have been reported for
seven species (
4-6,
8,
9,
11-13,
20-24,
27,
33,
34,
36,
37,
40,
42,
45). When the
identities of the ORF
with the sequences of the seven species were
compared, the ORF
had significantly higher sequence identity with GyrA
proteins
(56.3% ± 12.4%) than with ParC proteins (38.3% ± 2.1%)
(
P < 0.001).
Gram-negative bacteria exhibited higher
identities for GyrA than
did gram-positive bacteria (data not shown);
E. carotovora GyrA
was most closely related (76% identity).
Based on these results
and the results of mutational analysis
(described below), we identified
this ORF as the
gyrA coding
region of
V. parahaemolyticus.
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FIG. 2.
Comparison by alignment of V. parahaemolyticus (upper) and E. coli (lower) GyrA amino
acid sequences. Residue numbers are given for the sequence of V. parahaemolyticus. An asterisk indicates absence of a residue.
Identical residues are indicated by dashes. Arrows above the amino acid
sequences correspond to the orientations and positions of VGYR-1,
VGYR-2, and VGYR-4 degenerated primers (Table 1) used to amplify the
QRDR of V. parahaemolyticus GyrA.
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The
parC gene sequence contained a predicted ORF encoding
761 amino acids, the sequence of which was homologous with that
of
E. coli ParC (Fig.
3). The ORF
sequence was preceded by putative
promoter and ribosome binding
sequences (data not shown). The
amino acid sequence identities of this
ORF with ParC and GyrA
proteins of other bacterial species were
compared. This ORF sequence
was significantly more homologous with the
ParC sequences (49.4%
± 15.7% identity) than with the GyrA sequences
(37.2% ± 1.7% identity)
of the seven species for which both GyrA and
ParC sequences are
known (
P < 0.005). Gram-negative
bacteria exhibited higher identities
for ParC than did gram-positive
bacteria (data not shown);
E. coli ParC was most closely
related (69% identity). These results
and the results of mutational
analysis (described below) permitted
us to identify this ORF as the
parC coding region of
V. parahaemolyticus.
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FIG. 3.
Comparison by alignment of V. parahaemolyticus (upper) and E. coli (lower) ParC amino
acid sequences. Residue numbers are given for the sequence of V. parahaemolyticus. An asterisk indicates absence of a residue.
Identical residues are indicated by dashes. Arrows above the amino acid
sequences correspond to the orientations and positions of VGYR-1,
VGYR-3, and VGYR-4 degenerated primers (Table 1) used to amplify the
QRDR of V. parahaemolyticus ParC.
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Isolation and characterization of CIP-resistant mutants.
Mutants of AQ3815 that are resistant to CIP at various concentrations
were isolated in a stepwise manner, as shown in Table 2. These mutants were also resistant to
various other quinolone antibacterials (Table 2).
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TABLE 2.
Isolation of CIP-resistant mutants of V. parahaemolyticus AQ3815 and their susceptibilities to various
quinolone antibacterials
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The QRDRs of the
gyrA and
parC genes of AQ3815
and the CIP-resistant mutants were amplified by single-step PCR with
specific
primers (Table
1). The amplified sequences were cloned, and
the
nucleotide sequences were determined and compared. Mutations
detected
within the amplified regions of the mutant strains are
summarized
in Table
3. One base change in
the
gyrA sequence responsible
for a Ser-to-Ile change at
residue position 83 was detected in
all four mutant strains. In
addition, one base change in the
parC gene causing a
Ser-to-Phe change at residue position 85 was detected
in two of the
mutant strains that were resistant to high concentrations
of quinolones
(for VP-M3 and -M4, MIC of CIP was
50 µg/ml [Tables
2 and
3]).
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TABLE 3.
Mutations detected in the gyrA and
parC sequences of CIP-resistant mutants of V. parahaemolyticus AQ3815
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| |
DISCUSSION |
This study demonstrated that V. parahaemolyticus
carries nucleotide sequences highly homologous to the gyrA
and parC genes of other bacterial species and that these
sequences are mainly involved in experimentally induced resistance to
quinolone antibacterials. Based on these results, we designated these
sequences the gyrA and parC genes of V. parahaemolyticus. The approach that we used to determine the
nucleotide sequences of these genes is based on conservation of the
QRDR sequences and thus would be applicable to sequence determinations
of the gyrA and parC homologues in many bacterial species.
Mutant strains of AQ3815 that became resistant to CIP had a base
substitution at residue position 83 of GyrA or substitutions at both
position 83 of GyrA and position 85 of ParC (Table 3). Mutations in the
corresponding residues of GyrA and ParC in other bacterial species were
shown to be associated with quinolone resistance (30). The
stepwise appearance of a gyrA mutation followed by a
parC mutation in AQ3815 derivatives with increasing CIP
concentrations suggests that DNA gyrase and Topo IV may be the primary
and secondary targets of CIP, respectively, in V. parahaemolyticus. Similar observations were made for other
gram-negative bacteria, such as E. coli and N. gonorrhoeae (5, 15). Since no mutations other than the
above were found in the gyrA and parC QRDRs of the AQ3815-derived mutants, increases in the level of resistance from
VP-M1 to -M2 and from VP-M3 to -M4 must be explained by a mutation(s)
in non-QRDRs. Mutations in the gyrB gene or those that can
cause decreased drug permeability or increased drug efflux are possible
explanations (30). These possibilities need to be examined
in a future study. In any event, the use of quinolones should be
avoided as much as possible to prevent the emergence and spread of
quinolone-resistant strains of V. parahaemolyticus in the environment.
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ACKNOWLEDGMENTS |
This research was supported in part by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Science, Sports and
Culture, Japan.
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FOOTNOTES |
*
Corresponding author. Mailing address: 5 Nakauchi-cho,
Misasagi, Yamashina-ku, Kyoto, Japan. Phone: 81-75-595-4641. Fax:
81-75-595-4755. E-mail: nishino{at}mb.kyoto-phu.ac.jp.
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Antimicrobial Agents and Chemotherapy, May 1999, p. 1156-1162, Vol. 43, No. 5
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
