We describe two mutants of Mycoplasma hominis PG-21 which show resistance to 16-membered macrolides but susceptibility to lincosamides, obtained by in vitro exposure to increasing doses of josamycin. The 23S rRNA gene showed that each had a mutation (A2062G and A2062T) corresponding to nucleotide 2062 in Escherichia coli, which was associated with the acquired phenotype.The genetic bases for macrolide-lincosamide-streptogramin B group resistance have been extensively studied for many bacteria (2, 4-6, 16, 18, 27, 29, 30, 32-34) and mycoplasmas (8, 14). In Mycoplasma pneumoniae (14,22), as well as in other microorganisms, resistance to erythromycin has been associated with point mutations (A-to-G transition) in the loop of domain V of the 23S rRNA (2, 4-6, 8, 16, 18, 27, 29, 30, 32). Specific residues within domain V of 23S rRNA are involved in the action of macrolide-lincosamide-streptogramin antibiotics and chloramphenicol (2-6, 8, 11, 14-18, 20, 27-30, 32). It is well known that Mycoplasma hominis and many other mycoplasmas are resistant to erythromycin but susceptible to 16-membered macrolides (josamycin and miocamycin) (1, 7-9, 19, 23) and lincosamides (23). Recently the natural resistance of M. hominis to erythromycin has been associated with a guanine-to-adenine transition in position 2057 (Escherichia coli numbering), located in the central loop of the 23S rRNA domain V (8), and such a transition is present in Mycoplasma flocculare and Mycoplasma hyopneumoniae, which are also resistant to erythromycin (24).These features encouraged us to investigate the ability of josamycin to select for resistance in M. hominis strains, in order to see whether the acquired resistance pattern includes either macrolides only or both macrolides and lincosamides and to establish a possible relationship between the resistant phenotype and the appearance of specific point mutations in the region coding for the peptidyl transferase loop in the 23S rRNA gene.The type strain M. hominis PG-21 was chosen for our study. The strain was grown in SP-4 broth (pH 7.0) (7,8,21,25,26,31). The MIC was determined by a broth microdilution assay as previously described (7-9, 23).For multistep selection for resistance, SP-4 broth medium containing doubling concentrations of josamycin was inoculated with strain PG-21, incubated at 37°C, and examined for growth each day. To test for the development of resistance, 0.1 ml of the culture was withdrawn from each tube every day and spread on an SP-4 agar plate containing 1 g of josamycin/ml. All the colonies growing on josamycin-agar were challenged with increasing concentrations of the drug in broth (from 16 to 128 g/ml). All selected mutants were single colony purified on SP-4 agar-josamycin and drug-free SP-4 agar. Resistance to josamycin was assayed after the microrganisms were repeatedly transferred to antibiotic-free media.Two resistant clones of the M. hominis PG-21 strain were obtained by the selection procedure outlined above and were found to be stably resistant. The resistance/susceptibi...

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