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. 1999 May;43(5):1156–1162. doi: 10.1128/aac.43.5.1156

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, Takeshi Nishino 2,*
PMCID: PMC89126  PMID: 10223929

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.

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.

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 [α-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.

FIG. 1.

FIG. 1

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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.

TABLE 1.

Oligonucleotide primers used in this study

Primer Orientation when used as primer Positionsa Base sequence
VGYR-1 Sense GyrA, 41–46; ParC, 43–48 5′-T(A/C/G/T)AA(A/G)CC(A/C/G/T)GT(A/C/G/T)CA(C/T)(A/C)G-3′
VGYR-2 Sense GyrA, 57–62 5′-AA(C/T)GA(C/T)TGGAA(C/T)AA(A/G)(G/C)C-3′
VGYR-3 Sense ParC, 78–83 5′-AA(A/G)TA(C/T)CA(C/T)CC(A/C/G/T)CA(C/T)G-3′
VGYR-4 Antisense GyrA, 124–119; ParC, 127–122 5′-TC(A/C/G/T)GT(A/G)TA(A/C/G/T)C(G/T)CAT(A/C/G/T)GC-3′
GYRAS1 Antisense 564–534 5′-CGGTGTAACGCATTGCCGCAGCAGAGTCGCC-3′
GYRAS2 Antisense 395–365 5′-GGCTGATTTTTTATATGGTTTGTTCCAATCG-3′
GYRS1 Sense 365–395 5′-CGATTGGAACAAACCATATAAAAAATCAGCC-3′
GYRS2 Sense 534–564 5′-GGCGACTCTGCTGCGGCAATGCGTTACACCG-3′
PARAS1 Antisense 465–434 5′-GGAGCACCCCAGTTTCCCTGACCATCAACCAG-3′
PARAS2 Antisense 441–408 5′-TCAACCAGTGGGTAACGGTAAGAGAATGGTTGCG-3′
PARS1 Sense 403–436 5′-AATGGCGCAACCATTCTCTTACCGTTACCCACTG-3′
PARS2 Sense 434–465 5′-CTGGTTGATGGTCAGGGAAACTGGGGTGCTCC-3′
GYRCL1 Sense 11–42 5′-AAATATAGTCAGTTTCAGAAAGATATGGGATG-3′
GYRCL2 Antisense 2895–2864 5′-TGATGAAGATACAAAAAAGACGAGAATTTTGC-3′
PARCL1 Sense 11–40 5′-ATCGACGACTCTGAAGCCACTATGGAAATG-3′
PARCL2 Antisense 2523–2495 5′-GTTGGTATCATTCGTAAAGGCAAGGAATG-3′
GYRM1 Sense 365–386 5′-CGATTGGAACAAACCATATAAA-3′
GYRM2 Antisense 564–545 5′-CGGTGTAACGCATTGCCGCA-3′
PARM1 Sense 293–312 5′-CTTGGTCTTTCGGCATCAGC-3′
PARM2 Antisense 506–487 5′-CTTCGGTATAACGCATTGCC-3′
Cassette C1 5′-GTACATATTGTCGTTAGAACGCGTAATACGACTCA-3′
Cassette C2 5′-CGTTAGAACGCGTAATACGACTCACTATAGGGAGA-3′
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a

The positions of VGYR-1, VGYR-2, VGYR-3, and VGYR-4 degenerate primers correspond to amino acid residues of E. coli GyrA and ParC (Fig. 2 and Fig. 3). The positions of other primers correspond to base positions of the V. parahaemolyticus gyrA and parC genes deposited in GenBank (accession no. AB023569 and AB023570, respectively); primer designations starting with GYR and PAR indicate primers for the gyrA and parC genes, respectively. 

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 (Mg2+) (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 (Mg2+), 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 (Mg2+), 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 GeneTaq buffer, and 2.5 U of GeneTaq 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.

FIG. 2.

FIG. 2

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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.

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.

FIG. 3.

FIG. 3

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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.

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).

TABLE 2.

Isolation of CIP-resistant mutants of V. parahaemolyticus AQ3815 and their susceptibilities to various quinolone antibacterials

Strain Selection step Concn (μg/ml) of CIP used for isolation Isolation frequency MIC (μg/ml)a
CIP ENX NAD NOR OFX SPX NAL
AQ3815b nac na na 0.20 0.39 0.39 0.20 0.39 0.39 0.39
VP-M1 1st 0.78 6 × 10−8 0.78 3.13 3.13 0.78 0.78 0.39 12.5
VP-M2 2nd 3.13 4 × 10−7 6.25 25 6.25 25 6.25 3.13 50
VP-M3 3rd 25 6 × 10−8 50 100 25 100 50 12.5 100
VP-M4 4th 50 1 × 10−7 200 100 100 >200 200 50 100
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a

ENX, enoxacin; NAD, nadifloxacin; NOR, norfloxacin; OFX, ofloxacin; SPX, sparfloxacin; NAL, nalidixic acid. 

b

Wild-type strain. 

c

Not applicable. 

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]).

TABLE 3.

Mutations detected in the gyrA and parC sequences of CIP-resistant mutants of V. parahaemolyticus AQ3815

Strain MIC of CIP (μg/ml) Mutation in:
gyrA
parC
Amino acid residue
Base change Amino acid residue
Base change
Positiona Mutation Positiona Mutation
VP-M1 0.78 83 Ser→Ile AGG→ATT  —b — —
VP-M2 6.25 83 Ser→Ile AGG→ATT — — —
VP-M3 50 83 Ser→Ile AGG→ATT 85 Ser→Phe TCT→TTT
VP-M4 200 83 Ser→Ile AGG→ATT 85 Ser→Phe TCT→TTT
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a

Residue numbers counted from the N-terminal residue. 

b

—, no change. 

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.

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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