NpmC – a novel A1408 16S rRNA methyltransferase in the gut of humans and animals

NpmA and NpmB are 16S rRNA methyltransferases that act on residue A1408 and confer high-level resistance to almost all aminoglycosides; however, these methyltransferases are rarely reported. A novel gene, npmC, was identified after analysisng all world-wide available metagenomic projects in a One Health context. This gene has a high level of similarity (91.5%) with npmA and up to 92.7% similarity at amino acidic level. The protein encoded by this gene presents the conserved motifs required for A1408 methylation. npmC was synthesized and its expression in Escherichia coli resulted in a high level of resistance to 4,5-disubstituted 2-deoxystreptamine (2-DOS) and 4-monosubstituted 2-DOS aminoglycosides, as well as moderate resistance to 4,6-disusbstituted 2-DOS aminoglycosides, including the last resort aminoglycoside, plazomicin. Methylation at residue A1408 was confirmed by mass spectrometry assays. Analysis of the npmC gene background revealed that its genetic context was associated with different insertion sequences that could mobilise the gene. Similarities in the genetic context between npmC and npmA indicate that they share a common ancestor. The immediate genetic context of this methyltransferase indicates a high relationship to the Eubacteriales order. This finding reveals the dark matter of the microbiome as a potential source of novel resistance genes, expands the list of the true pan-aminoglycoside 16S rRNA methyltransferases, which threaten the usefulness and development of next-generation aminoglycosides.