Multiplex amplicon sequencing reveals polyclonality and low macrolide resistance in the French Mycoplasma pneumoniae outbreak, 2023–2024

Abstract Objectives Unprecedented global increases in Mycoplasma pneumoniae respiratory infections began in late summer 2023. Although a predominance of p1 subtype 1 associated with alarmingly high rates of macrolide resistance was reported in Southeast Asia, data from Europe remain limited. We aimed to characterise the circulating M. pneumoniae strains in French hospitalised patients, using a high-throughput amplicon sequencing approach. Methods Phylogenetic analysis was performed using p1 adhesin gene subtyping and multi-locus sequence typing. Mutations associated with antibiotic resistance were screened in the 23S and 16S rRNA, rplD, rplV, gyrA, and parC genes. Results Although most patients (835/933; 89.5%) presented with respiratory symptoms, extrapulmonary manifestations were observed in 49/884 (5.5%) patients. Five p1 subtypes and 29 distinct sequence types were identified, with a predominance of p1 subtype 2 (67.2%) and ST14 (29.7%). Subtype 2 was more frequent in adults, whereas subtype 1 was more often associated with extrapulmonary manifestations. Macrolide resistance-associated mutations were observed at a 3.0% prevalence, exhibiting a trend toward subtype 1 and high frequency of mixed populations. No mutations clearly associated with fluoroquinolone or tetracycline resistance were identified. Conclusions This study demonstrates the polyclonal spread of M. pneumoniae strains and a low prevalence of macrolide resistance during the 2023–2024 outbreak in France.