Abstract
Mycoplasma pneumoniae is a significant cause of community-acquired pneumonia, with epidemic peaks occurring every 3–7 years. This multicenter, retrospective study analyzed 421 M. pneumoniae strains from four regions in China (Liaoning, Zhejiang, Wuhan, Guizhou) collected between November 2023 and January 2024, alongside 147 publicly available genomes, to investigate genotypic diversity and macrolide resistance during a recent resurgence. Infection rates in 2023 ranged from 19.4 to 35.9%, reflecting a significant increase compared to the reduced incidence (1.69%) during the COVID-19 pandemic (2020–2021). Genomic sequencing revealed that the P1-1 genotype predominated (87.3–94.9%), with P1-2c and a rare P1-2 g variant also detected. Macrolide resistance was observed in 97.1% of samples, primarily driven by the A2063G mutation in the 23 S rRNA gene, though 80% of P1-2 strains lacked resistance mutations. Phylogenetic analysis, including sequences from Beijing, Shenzhen, Suzhou, and Taiwan, indicated widespread circulation of macrolide-resistant P1-1 strains since 2016. No significant differences in bacterial load or blood parameters were observed between genotypes or resistance profiles. The resurgence aligns with global trends, with P1-1 dominance and high macrolide resistance complicating treatment, particularly in children. These findings highlight the need for ongoing surveillance, updated treatment guidelines, and alternative therapeutic strategies to address the challenges posed by macrolide-resistant M. pneumoniae. Continuous monitoring of genotypic variations and resistance patterns is essential to inform public health strategies and improve clinical outcomes. Supplementary Information: The online version contains supplementary material available at 10.1038/s41598-025-29135-7.