Induction of macrolide resistance in Mycoplasma gallisepticum in vitro and its resistance-related mutations within domain V of 23S rRNA.

Antibiotic-resistant mutants of Mycoplasma gallisepticum were selected in vitro from the susceptible strains S6 and BG44T by serial passages in stepwise concentrations of erythromycin, tylosin, or tilmicosin. High resistance to erythromycin or tilmicosin developed readily, whereas resistance to tylosin developed only after greater numbers of passages. Three mutants selected by each selector antibiotic were cloned and detected, and all cloned mutants exhibited cross-resistance to the three selector antibiotics as well as to lincomycin. Portions of the genes encoding domain V of 23S rRNA of the cloned mutants were amplified by PCR, and their nucleotide sequences were compared to those of the susceptible parent strains. Five of the six mutants selected by erythromycin harbored an A2058G (Escherichia coli numbering) mutation in one of the two 23S rRNA. One of the six mutants selected by erythromycin harbored a G2057A mutation and an A2059G mutation in the other 23S rRNA. In tilmicosin-selected mutants, two mutations, A2058G and A2503U, occurred in one of the two 23S rRNA. No mutation was detected in the two 23S rRNA of tylosin-selected mutants with low-level resistance. Mutations at homologous locations in the 23S rRNA of other macrolide-resistant bacteria indicate that the phenotype of macrolide resistance occurring in M. gallisepticum is strongly associated with point mutations in domain V of 23S rRNA.