Alterations in topoisomerase IV and DNA gyrase in quinolone-resistant mutants of Mycoplasma hominis obtained in vitro

Abstract

Mycoplasma hominis mutants were selected stepwise for resistance to ofloxacin and sparfloxacin, and their gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized. For ofloxacin, four rounds of selection yielded six first-, six second-, five third-, and two fourth-step mutants. The first-step mutants harbored a single Asp426-->Asn substitution in ParE. GyrA changes (Ser83-->Leu or Trp) were found only from the third round of selection. With sparfloxacin, three rounds of selection generated 4 first-, 7 second-, and 10 third-step mutants. In contrast to ofloxacin resistance, GyrA mutations (Ser83-->Leu or Ser84-->Trp) were detected in the first-step mutants prior to ParC changes (Glu84-->Lys), which appeared only after the second round of selection. Further analysis of eight multistep-selected mutants of M. hominis that were previously described (2) revealed that they carried mutations in ParE (Asp426-->Asn), GyrA (Ser83-->Leu) and ParE (Asp426-->Asn), GyrA (Ser83-->Leu) and ParC (Ser80-->Ile), or ParC (Ser80-->Ile) alone, depending on the fluoroquinolone used for selection, i.e., ciprofloxacin, norfloxacin, ofloxacin, or pefloxacin, respectively. These data indicate that in M. hominis DNA gyrase is the primary target of sparfloxacin whereas topoisomerase IV is the primary target of pefloxacin, ofloxacin, and ciprofloxacin.

FIG. 1

Nucleotide and amino acid sequences of the MH8-MH10-amplified DNA fragment of the parC gene (A) and the MH28-MH29-amplified DNA fragment of the parE gene (B) of M. hominis. The region corresponding to the gyrA QRDR of E. coli is underlined (A). Asterisks indicate the mutations found in quinolone-resistant mutants of M. hominis.

FIG. 2

Alignment of the M. hominis (Mh) ParC (A) and ParE (B) sequences with those of M. genitalium (Mg) (10), M. pneumoniae (Mp) (16), B. subtilis (Bs) (27), S. aureus (Sa) (9), S. pneumoniae (Sp) (24), and E. coli (Ec) (18). The sequence of M. gallisepticum (Mga) topoisomerase II subunit B (28) also is included in comparisons in panel B. The arrow in panel A indicates the Tyr residue involved in DNA binding. The region corresponding to the E. coli GyrA QRDR (residues 67 to 106) (A) and positions 426 and 447, corresponding to the E. coli GyrB QRDR (B), are indicated. The ParE conserved motifs VEGDSAGG and PL(R/K)GK are underlined. Residues involved in quinolone resistance in M. hominis are underlined and in boldface type. Asterisks indicate identical amino acids, and dashes indicate gaps introduced to maximize similarities. Percentages of identical amino acids are in parentheses.

FIG. 3

Relationships among M. hominis PG21 and fluoroquinolone-resistant mutants IO1 to IVO5, selected by stepwise exposure to ofloxacin. First-, second-, third-, and fourth-step mutants are designated by the prefixs I, II, III, and IV, respectively. The numbers outside the boxes indicate the OFX concentrations (in micrograms per milliliter) used in the selection steps. The superscripts ⁺, *, and ° indicate the presence of mutations in parE, gyrA, and parC, respectively (see Table 2).

FIG. 4

Relationships among M. hominis PG21 and fluoroquinolone-resistant mutants IS1 to IIIS4B, selected by stepwise exposure to SFX. First-, second-, and third-step mutants are designated by the prefixes I, II, and III, respectively. The numbers outside the boxes indicate the SFX concentrations (in micrograms per milliliter) used in the selection steps. The superscripts * and ° indicate the presence of mutations in gyrA and parC, respectively (see Table 3).