16S rRNA Gene Mutations Associated with Decreased Susceptibility to Tetracycline in Mycoplasma bovis

Mycoplasma bovis isolates with decreased susceptibility to tetracyclines are increasingly reported worldwide. The acquired molecular mechanisms associated with this phenomenon were investigated in 70 clinical isolates of M. bovis. Sequence analysis of the two 16S rRNA-encoding genes (rrs3 and rrs4 alleles) containing the primary binding pocket for tetracycline (Tet-1 site) was performed on isolates with tetracycline hydrochloride minimal inhibitory concentrations of 0.125-16 μg/ml. Mutations at positions A965T, A967T/C (Escherichia coli numbering) of helix 31, at position U1199C of helix 34 and at position G1058A/C were identified. Decreased susceptibility to tetracycline (MICs of ≥ 2 μg/ml) was associated with mutations present at two (A965 and A967) or three positions (A965, A967 and G1058) of both rrs alleles. No tet(M), tet(O) or tet(L) determinants were found in the genome of any of 70 M. bovis isolates. The data presented correlates (P < 0.0001) the mutations identified in the Tet-1 site of clinical isolates of M. bovis with decreased susceptibility to tetracycline.