Evolution and characteristics of tetracycline resistance in <i>Mycoplasma pneumoniae</i>

Macrolide resistance in <i>Mycoplasma pneumoniae</i> is becoming prevalent. Tetracyclines are an important alternative for patients with macrolide-resistant <i>M. pneumoniae</i> infections. Tetracycline-resistant <i>M. pneumoniae</i> has never been reported in clinical isolates or <i>in vitro</i>-induced strains. Here, we aimed to explore tetracycline-resistant <i>M. pneumoniae in vitro</i> and the underlying resistance mechanisms. Clinically isolated parental <i>M. pneumoniae</i> strains were selected for tetracycline resistance by inducing them <i>in vitro</i> with increasing concentrations of tetracycline, minocycline, and tigecycline. The whole genomes of the induced resistant and parent strains were both sequenced to identify tetracycline-resistance genes, and their 16S rRNA gene sequences were compared. Ribosome-binding assays were conducted by incubating the 30S ribosomes of the resistant and sensitive strains with various concentrations of ³H-labeled tetracycline. The effect of efflux pump inhibitor reserpine on the susceptibility of tetracyclines to the resistant mutants was evaluated. Eight resistant mutants were obtained from 12 tetracycline-susceptible <i>M. pneumoniae</i> strains. Whole-genome sequencing identified point mutations in the 16S rRNA gene of all the resistant mutants. The ribosomes of two induced resistant strains showed lower tetracycline-binding capacities than their parental strains. Reserpine increased the susceptibility of two resistant mutants to tetracyclines by more than fourfold. None of the resistant mutants carried the known tetracycline-resistance genes. <i>M. pneumoniae</i> strains can develop tetracycline resistance. Mutations in the 16S rRNA gene leading to decreased ribosomal-binding capacity and the presence of efflux pumps may be the mechanisms associated with tetracycline resistance in <i>M. pneumoniae</i>. This study identifies markers to track the emergence of tetracycline resistance in <i>M. pneumoniae</i>.IMPORTANCEResistance to anti-<i>Mycoplasma pneumoniae</i> antibiotics is a global public health problem. Effective antibiotic treatment strategies are urgently needed. Tetracyclines are among the most commonly used antibiotics for the treatment of drug-resistant <i>M. pneumoniae</i> infections. Tetracycline-resistant <i>M. pneumoniae</i> has not been reported; however, treatment with increased concentrations of tetracycline selects hyposensitive mutants. In this study, we successfully induced tetracycline-resistant <i>M. pneumoniae</i> mutants by 10-month <i>in vitro</i> serial passage experiments and found that a 16S rRNA mutation resulting in reduced ribosomal drug binding was the mechanism that led to tetracycline resistance in <i>M. pneumoniae</i>. In addition, the efflux pump might also be related to decreased tetracycline susceptibility. This study presented the first data on the induction of tetracycline resistance in <i>M. pneumoniae in vitro</i>, providing insights into the development of effective treatment strategies for drug-resistant <i>M. pneumoniae</i> infections in the future.