Sequence analysis of the gyrA and parC homologues of a wild-type strain ofVibrio parahaemolyticus and its fluoroquinolone-resistant mutants

Vibrio parahaemolyticus causes seafood-borne gastroenteritis in humans. It is particularly important in Japan, where raw seafood is frequently consume d. Fluoroquinolone is one of the current drugs of choice for treating patie nts infected by V. parahaemolyticus because resistant strains are rarely fo und. To study a possible fluoroquiuolone resistance mechanism in this organ ism, nucleotide sequences that are homologous to known gyrA and parC genes have been cloned from IT. parahaemolyticus AQ3815 and sequenced by amplific ation with degenerate primers of the quinolone resistance-determining regio n (QRDR), followed by cassette ligation-mediated PCR Open reading frames en coding polypeptides of 878 and 761 amino acid residues were detected in the gyrA and parC homologues, respectively. The V. parahaemolyticus GyrA and P arC sequences were most closely related to Erwinia carotovora GyrA (76% ide ntity) and Escherichia coli ParC (69% identity) sequences, respectively. Ci profloxacin-resistant mutants of AQ3815 were obtained on an agar medium by multistep selection with increasing levels of the quinolone. One point muta tion only in the gyrA QRDR was detected among mutants with low- to intermed iate-level resistance, while point mutations in both the gyrA and parC QRDR s were detected only in strains with high-level resistance. These results s trongly suggest that, as in other gram-negative bacteria, GyrA and ParC are the primary and secondary targets, respectively, of ciprofloxacin in V. pa rahaemolyticus.