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The macrolide class of antibiotics, including tylosin and tilmicosin, is widely used in the veterinary field for prophylaxis and treatment of mycoplasmosis. In vitro susceptibility testing of 50 strains of M. gallisepticum isolated in Israel during the period 1997-2010 revealed that acquired resistance to tylosin as well as to tilmicosin was present in 50% of them. Moreover, 72% (13/18) of the strains isolated from clinical samples since 2006 showed acquired resistance to enrofloxacin, tylosin and tilmicosin. Molecular typing of the field isolates, performed by gene-target sequencing (GTS), detected 13 molecular types (I-XIII). Type II was the predominant type prior to 2006 whereas type X, first detected in 2008, is currently prevalent. All ten type X strains were resistant to both fluoroquinolones and macrolides, suggesting selective pressure leading to clonal dissemination of resistance. However, this was not a unique event since resistant strains with other GTS molecular types were also found. Concurrently, the molecular basis for macrolide resistance in M. gallisepticum was identified. Our results revealed a clear-cut correlation between single point mutations A2058G or A2059G in domain V of the gene encoding 23S rRNA (rrnA, MGA_01) and acquired macrolide resistance in M. gallisepticum. Indeed, all isolates with MIC ≥ 0.63 μg/mL to tylosin and with MIC ≥ 1.25 μg/mL to tilmicosin possess one of these mutations, suggesting an essential role in decreased susceptibility of M. gallisepticum to 16-membered macrolides.

The mechanism responsible for acquired decreased susceptibility to macrolides (14-membered erythromycin [Ery], 16-membered tylosin [Ty] and tilmicosin [Tm]) and to lincosamides (lincomycin [Ln]) was investigated in Mycoplasma synoviae, a pathogen that causes respiratory infections and synovitis in chicken and turkey. Sequence analysis of domains II and V of the two 23S rRNA alleles and ribosomal proteins L4 and L22 was performed on 49 M. synoviae isolates, M. synoviae type strain WVU1853, and reference strain FMT showing minimal inhibitory concentrations (MICs) to Ty (≤ 0.015 to 2 μg/ml), Tm (0.03 to ≥ 8 μg/ml), and Ln (0.125 to 8 μg/ml); MICs to Ery ranged from 32 to ≥ 128 μg/ml. Our results showed that the nucleotide substitution G748A (Escherichia coli numbering) in domain II of one or both 23S rRNA alleles may account for a slight increase in MICs to Ty and Tm (up to 0.5 and 2 μg/ml, respectively). No correlation between the presence of G748A and decreased susceptibility to Ln was found. However, the presence of the point mutations A2058G or A2059G in domain V of one or both alleles of the 23S rRNAs was correlated with a more significant decrease in susceptibility to Ty (1-2 μg/ml), Tm (≥ 8 μg/ml), and Ln (≥ 8 μg/ml). All M. synoviae isolates tested had a G2057A transition in the 23S rRNAs consistent with previously described intrinsic resistance to Ery. Mutations G64E (one isolate) and Q90K/H (two isolates) were identified in the L4 and L22 proteins, respectively, but their impact on decreased susceptibility to macrolides and lincomycin was not clear.