Type II topoisomerase quinolone resistance-determining regions of Aeromonas caviae, A. hydrophila, and A. sobria complexes and mutations associated with quinolone resistance

Abstract

Most Aeromonas strains isolated from two European rivers were previously found to be resistant to nalidixic acid. In order to elucidate the mechanism of this resistance, 20 strains of Aeromonas caviae (n = 10), A. hydrophila (n = 5), and A. sobria (n = 5) complexes, including 3 reference strains and 17 environmental isolates, were investigated. Fragments of the gyrA, gyrB, parC, and parE genes encompassing the quinolone resistance-determining regions (QRDRs) were amplified by PCR and sequenced. Results obtained for the six sensitive strains showed that the GyrA, GyrB, ParC, and ParE QRDR fragments of Aeromonas spp. were highly conserved (> or =96.1% identity), despite some genetic polymorphism; they were most closely related to those of Vibrio spp., Pseudomonas spp., and members of the family Enterobacteriaceae (72.4 to 97.1% homology). All 14 environmental resistant strains carried a point mutation in the GyrA QRDR at codon 83, leading to the substitution Ser-83-->Ile (10 strains) or Ser-83-->Arg. In addition, seven strains harbored a mutation in the ParC QRDR either at position 80 (five strains), generating a Ser-80-->Ile (three strains) or Ser-80-->Arg change, or at position 84, yielding a Glu-84-->Lys modification. No amino acid alterations were discovered in the GyrB and ParE QRDRs. Double gyrA-parC missense mutations were associated with higher levels of quinolone resistance compared with the levels associated with single gyrA mutations. The most resistant strains probably had an additional mechanism(s) of resistance, such as decreased accumulation of the drugs. Our data suggest that, in mesophilic Aeromonas spp., as in other gram-negative bacteria, gyrase and topoisomerase IV are the primary and secondary targets for quinolones, respectively.

FIG.1.

Nucleotide sequences of gyrA (a), gyrB (b), parC (c), and parE (d) fragments containing the QRDRs of Aeromonas spp. The sequences of the six sensitive strains of A. caviae (Ac), A. hydrophila (Ah), and A. sobria (As) are as determined in the present study; Ac7616, Ah7614, and As7433 are the reference CIP strains. The A. salmonicida (A. sal) gyrA sequence is as reported by Oppegaard and Sørum (30). The E. coli numbering system is used (GenBank accession numbers are given in footnote c of Table 1). QRDRs are framed and shaded. Nucleotide differences are indicated with boldface characters and are underlined when the difference led to amino acid changes. The corresponding nucleotide (Nt.) and proteic (Pr.) consensus sequences are shown.

FIG.1.

Nucleotide sequences of gyrA (a), gyrB (b), parC (c), and parE (d) fragments containing the QRDRs of Aeromonas spp. The sequences of the six sensitive strains of A. caviae (Ac), A. hydrophila (Ah), and A. sobria (As) are as determined in the present study; Ac7616, Ah7614, and As7433 are the reference CIP strains. The A. salmonicida (A. sal) gyrA sequence is as reported by Oppegaard and Sørum (30). The E. coli numbering system is used (GenBank accession numbers are given in footnote c of Table 1). QRDRs are framed and shaded. Nucleotide differences are indicated with boldface characters and are underlined when the difference led to amino acid changes. The corresponding nucleotide (Nt.) and proteic (Pr.) consensus sequences are shown.

FIG.1.

Nucleotide sequences of gyrA (a), gyrB (b), parC (c), and parE (d) fragments containing the QRDRs of Aeromonas spp. The sequences of the six sensitive strains of A. caviae (Ac), A. hydrophila (Ah), and A. sobria (As) are as determined in the present study; Ac7616, Ah7614, and As7433 are the reference CIP strains. The A. salmonicida (A. sal) gyrA sequence is as reported by Oppegaard and Sørum (30). The E. coli numbering system is used (GenBank accession numbers are given in footnote c of Table 1). QRDRs are framed and shaded. Nucleotide differences are indicated with boldface characters and are underlined when the difference led to amino acid changes. The corresponding nucleotide (Nt.) and proteic (Pr.) consensus sequences are shown.

FIG.1.

Nucleotide sequences of gyrA (a), gyrB (b), parC (c), and parE (d) fragments containing the QRDRs of Aeromonas spp. The sequences of the six sensitive strains of A. caviae (Ac), A. hydrophila (Ah), and A. sobria (As) are as determined in the present study; Ac7616, Ah7614, and As7433 are the reference CIP strains. The A. salmonicida (A. sal) gyrA sequence is as reported by Oppegaard and Sørum (30). The E. coli numbering system is used (GenBank accession numbers are given in footnote c of Table 1). QRDRs are framed and shaded. Nucleotide differences are indicated with boldface characters and are underlined when the difference led to amino acid changes. The corresponding nucleotide (Nt.) and proteic (Pr.) consensus sequences are shown.