Cloning and nucleotide sequence of the DNA gyrase (gyrA) gene from Mycoplasma hominis and characterization of quinolone-resistant mutants selected invitro with trovafloxacin

We report the cloning and characterization of the gyrA gene of the Mycoplas ma hominis DNA gyrase, which was previously shown to be associated with qui nolone resistance in this organism. The 2,733-bp gyrA gene encodes a protei n of 911 amino acids with a calculated molecular mass of 102.5 kDa. As expe cted, M. hominis GyrA exhibits higher homology with the GyrA subunits of th e gram-positive bacteria Clostridium acetobutylicum, Bacillus subtilis, Str eptococcus pneumoniae, and Staphylococcus aureus than with its Escherichia coli counterpart. Knowing the entire sequence of the gyrA gene of M. homini s could be very useful for confirming the role of the GyrA subunit in fluor oquinolone resistance. Twenty-nine mutants of M. hominis were selected step wise for resistance to trovafloxacin, a new potent fluoroquinolone, and the ir gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized. Three rounds of selection yielded 3 first-step, 12 second-s tep, and 14 third-step mutants. The first-step mutants harbored a single su bstitution, Glu460-->Lys (E. coli coordinates), in ParE. GyrA changes, Ser8 3-->Leu, Glu87-->Lys, and Ala119-->Glu or Val, were found only in the secon d round of selection. At the third step, additional substitutions, at ParC Ser80, Ser81, and Glu84 and ParE Leu440, associated with high-level resista nce to fluoroquinolones, appeared. Thus, high-level resistance to trovaflox acin required three steps and was associated with alterations in both fluor oquinolone targets. According to these genetic data, in M. hominis, as in S taphylococcus aureus and Streptococcus pneumoniae, topoisomerase IV seems t o be the primary target of trovafloxacin.