Evolution and characteristics of tetracycline resistance in Mycoplasma pneumoniae

Macrolide resistance in Mycoplasma pneumoniae is becoming prevalent. Tetracyclines are an important alternative for patients with macrolide-resistant M. pneumoniae infections. Tetracycline-resistant M. pneumoniae has never been reported in clinical isolates or in vitro-induced strains. Here, we aimed to explore tetracycline-resistant M. pneumoniae in vitro and the underlying resistance mechanisms. Clinically isolated parental M. pneumoniae strains were selected for tetracycline resistance by inducing them in vitro 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 M. pneumoniae 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. M. pneumoniae 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 M. pneumoniae. This study identifies markers to track the emergence of tetracycline resistance in M. pneumoniae.IMPORTANCEResistance to anti-Mycoplasma pneumoniae 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 M. pneumoniae infections. Tetracycline-resistant M. pneumoniae has not been reported; however, treatment with increased concentrations of tetracycline selects hyposensitive mutants. In this study, we successfully induced tetracycline-resistant M. pneumoniae mutants by 10-month in vitro 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 M. pneumoniae. 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 M. pneumoniae in vitro, providing insights into the development of effective treatment strategies for drug-resistant M. pneumoniae infections in the future.