exclude mutants with one or two mutated genes and thereafter
screened high concentration resistant mutants among them. Among
38 mutants resistant to erythromycin at 0.1 μg/mL, 15 mutants were
found to be resistant to a higher concentration (250 to 5000 μg/mL) by
the second step of screenings. Nucleotide sequences of three copies of
23S ribosomal RNA genes were determined for these 15 mutants and
compared with that of B. bifidum YIT 4001. Eleven mutants had G
residue at the 2090th position and three had G at the 2091st position
of all three copies of 23S ribosomal RNA genes. However, one mutant
had G at the 2091st position of two out of three copies of 23S ribosomal
RNA genes (Table 2). According to E. coli numbering system, these
positions are equivalent to the 2058th and 2059th on the domain V of
23S ribosomal RNA which have been confirmed to determine
erythromycin resistance in many bacteria (Biswas et al., 2007; Ladely
et al., 2009). Although nucleotide residues are different between B.
bifidum YIT 4007 and newly isolated mutants, they were located at the
same position of 23S ribosomal RNA genes. Thus, it was likely that the
A2090C replacement is responsible for erythromycin resistance of B.
bifidum YIT 4007.