Genomic Characterization of 16S rRNA Methyltransferase-Producing Reveals the Emergence of ST6260 Harboring , , , and Genes in a Cancer Hospital in Bulgaria.

Acquired 16S rRNA methyltransferases (16S-RMTases) confer high-level resistance to aminoglycosides and are often associated with β-lactam and quinolone resistance determinants. Using PCR, whole-genome sequencing and conjugation experiments, we conducted a retrospective genomic surveillance study of 16S-RMTase-producing , collected between 2006 and 2023, to explore transmission dynamics of methyltransferase and associated antibiotic resistance genes. Among the 10,731 consecutive isolates, 150 (1.4%) from 13 species carried (92.7%), (4.7%), and + (2.7%) methyltransferase genes. The coexistence of extended-spectrum β-lactamase (, , ), carbapenemase (, , ), acquired AmpC (, , ), and plasmid-mediated quinolone resistance (, , ) genes within these isolates was also detected. Methyltransferase genes were carried by different plasmids (IncL/M, IncA/C, IncR, IncFIB, and IncFII), suggesting diverse origins and sources of acquisition. was co-transferred with , , , , , , , and , while was co-transferred with , highlighting the multidrug-resistant nature of these plasmids. Long-read sequencing of ST6260 isolates revealed a novel resistance association, with and on the chromosome, on a conjugative ColKP3 plasmid, and with on self-transmissible IncFIB and IncFII plasmids. The genetic plasticity of plasmids carrying methyltransferase genes suggests their potential to acquire additional resistance genes, turning 16S-RMTase-producing into a persistent public health threat.