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

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.