BACKGROUND: The resurgence of Mycoplasma pneumoniae (MP), first reported in China in 2023 was attributed to waning post-pandemic immunity with notable increases in macrolide-resistant MP (MRMP) (>80%). In Australia, infections peaked in early 2024, particularly among children under 15. While MRMP remains low in Europe, North America, and Australia (<5%), limited routine testing and surveillance restricts understanding of resistance dynamics. As macrolides are first-line therapy in many health settings, MRMP surveillance is essential for guiding empirical treatment and stewardship.

METHODS: We applied a novel capture-based targeted metagenomic sequencing (tNGS) to PCR-positive MP specimens (n=356) from across Australia. This approach enabled whole-genome recovery and MRMP detection directly from clinical specimens, without culture. MRMP detections were benchmarked against RT-PCR and clinical data were analysed to assess associations between resistance and healthcare utilisation.

RESULTS: This is the first genomics-informed national study of MP in Australia. We recovered 124 high-quality genomes, revealing a genetically diverse population with co-circulation of P1 Type 1 (69%) and Type 2 (31%). MRMP was identified in 13% of genomes, all belonging to clades prior to 2024 had only been reported in Asia (ST3 and ST14). MRMP cases were geographically widespread, suggesting importation and local transmission. Unlike reports from China, macrolide-susceptible clades (ST3, ST7, ST17 and ST20) predominated (87%) and were associated to significant lower healthcare utilisation compared to MRMP cases.

CONCLUSION: Our findings demonstrate the utility of tNGS for genomic epidemiology and highlight the need for MRMP surveillance. Although macrolides remain effective in Australia, emerging MRMP strains require close monitoring to inform treatment guidelines and antimicrobial stewardship.