Identification and characteristics of a novel acquired aminoglycoside phosphotransferase, APH(3′)-IVb, from <i>Riemerella anatipestifer</i>

A novel acquired aminoglycoside resistance gene, <i>aph(3</i>'<i>)-IVb</i>, was identified via whole-genome sequencing of a multidrug-resistant <i>Riemerella anatipestifer</i> isolate from a duck. The gene encodes a 262-amino-acid phosphotransferase, APH(3')-IVb, sharing only 39.9% amino acid identity with its closest known homolog, APH(3')-IVa. Heterologous expression of <i>aph(3</i>'<i>)-IVb</i> in <i>Escherichia coli</i> and a susceptible <i>R. anatipestifer</i> strain conferred resistance to neomycin, paromomycin, and ribostamycin, a phenotype validated by gene deletion and complementation experiments. Kinetic analysis of the purified APH(3')-IVb enzyme confirmed phosphotransferase activity against these three aminoglycosides, with catalytic efficiencies (<i>k</i>_cat/<i>K_m</i>) ranging from 10⁴ to 10⁵ M⁻¹·s⁻¹. Furthermore, site-directed mutagenesis identified key residues critical for enzymatic function. While the prevalence of <i>aph(3</i>'<i>)-IVb</i> in <i>R. anatipestifer</i> isolates was low (1.6%), analysis of public databases identified 93 <i>aph(3</i>'<i>)-IVb</i>-positive sequences, of which 36.6% originated from human pathogens. Genetic environment analysis revealed that <i>aph(3</i>'<i>)-IVb</i> resides within a genomic resistance island flanked by mobile genetic elements, suggesting its horizontal acquisition. The emergence of this novel enzyme, coupled with its association with mobile elements and distribution among human pathogens, underscores a potential pathway for resistance dissemination across veterinary and clinical environments, posing a significant public health concern.