Whole-genome probe capture sequencing reveals genomic diversity and characteristics of Mycoplasma pneumoniae in Nanjing, China

<i>Mycoplasma pneumoniae</i> (<i>M. pneumoniae</i>), a slow-growing, fastidious Gram-negative bacterium and a leading cause of community-acquired pneumonia globally, remains understudied and underreported across numerous geographical areas in China despite its worldwide significance. This study employed probe capture sequencing for targeted enrichment and direct sequencing of <i>M. pneumoniae</i> from clinical samples, combined with comparative genomic analyses of contemporary and historical global genomes. Core genome and pan-genome revealed that the <i>M. pneumoniae</i> genomes were classified into two distinct clades, P1-I and P1-II, each associated with a specific sequence type (ST). Most of the genomes sequenced in this study were identified as P1-I (86.96%, 20/23), contrasting with the previously reported predominance of P1-II in the area. A limited number of single-nucleotide variations were identified in the virulence-associated genes between P1-I and P1-II, leading to amino acid substitutions. The A2063G point mutation in the 23S rRNA gene was detected in all sequenced genomes (23/23), demonstrating a 100% mutation rate. This study provides the first reported application of probe capture methodology for <i>M. pneumoniae</i>, highlighting the critical importance of sustained surveillance efforts to monitor the evolution and epidemiology of this pathogen.