The parental strains PG31 and S6 were included in each MIC test as a control. A portion of the gene encoding domain V of 23S rRNA gene and the entire gene encoding ribosome protein L3 were amplified by PCR with specific primers. The primers were designed from the complete genome sequence of M. gallisepticum strain A5969 (GenBank accession no. AE015450). Because M. gallisepticum possesses two copies of the 23S rRNA gene (Chen & Finch, 1989), two pairs of primers (primer

pair MG23A-F and MG23A-R and primer pair MG23B-F and MG23B-R) were designed to amplify each 23S rRNA gene independently. Two internal primers, MGF-1879 and MGR-2763, were used to amplify domain V of 23S rRNA gene. Amplification of the entire gene of ribosomal protein L3 was performed with the primer pair L3-F and L3-R. The primers and reaction conditions are shown in Table 1. PCR products were purified using the AG-014699 mouse QIAquick Gel Extraction Kit (Qiagen) and sequenced using the same primers as those used for PCR. Random amplified polymorphic DNA (RAPD) analysis was performed, as described previously (Pakpinyo & Sasipreeyajan, 2007), to confirm that the mutants were derived from the corresponding parental strain. Mycoplasma gallisepticum mutants

with decreased susceptibility to pleuromutilins could be selected by serial passages of the parental strains M. gallisepticum S6 and PG31 in subinhibitory concentrations of tiamulin or valnemulin. INCB024360 mouse For the purposes of this study, we defined that the mutant exhibits resistance when the MIC increased ≥8-fold in comparison with the MIC obtained for the corresponding parental strain. Three subcultured clones from the passage with significantly increased MIC were studied. MICs of only one clone are shown in Table 2 because no significant difference was observed between the three clones. The resistance phenotype of all mutants was stable after five consecutive subcultures in an antibiotic-free

medium. Smoothened Moreover, the RAPD experiments showed that the profiles of the mutants were identical to the profile of the corresponding parental strain (data not shown). For the mutants, the MICs of tiamulin ranged from 0.5 to 64 μg mL−1, and the MICs of valnemulin ranged from 0.032 to 32 μg mL−1. The concentrations of valnemulin required to inhibit each acquired mutant were significantly lower than those for tiamulin (Table 2). Two susceptible strains PG31 and S6 were used for the selection. Although 10 passages were performed for both parental strains, the results of selection showed marked differences between the strains PG31 and S6. The highest tiamulin MIC for the mutants derived from PG31 was 16 μg mL−1, compared with 64 μg mL−1 for the mutants derived from S6, and the highest valnemulin MIC for the mutants derived from PG31 was 0.25 μg mL−1, compared with 32 μg mL−1 for the mutants derived from S6.