ABSTRACTAminoglycoside-modifying enzymes are among the most important mechanisms of resistance to aminoglycoside antibiotics, typically conferring high-level resistance by enzymatic drug inactivation. Previously, we isolated a multidrug-resistantBrucella intermediastrain ZJ499 from a cancer patient, and whole-genome sequencing revealed several putative novel aminoglycoside-modifying enzyme genes in this strain. Here, we report the characterization of one of them that encodes an intrinsic, chromosomal aminoglycoside nucleotidyltransferase designated ANT(9)-Ic, which shares only 33.05% to 47.44% amino acid identity with the most closely related ANT(9)-I enzymes. When expressed inEscherichia coli, ANT(9)-Ic conferred resistance only to spectinomycin and not to any other aminoglycosides tested, indicating a substrate profile typical of ANT(9)-I enzymes. Consistent with this, deletion ofant(9)-Icin ZJ499 resulted in a specific and significant decrease in MIC of spectinomycin. Furthermore, the purified ANT(9)-Ic protein showed stringent substrate specificity for spectinomycin with aKmvalue of 44.83 μM and akcat/Kmof 2.8 × 104M−1s−1, echoing the above observations of susceptibility testing. In addition, comparative genomic analysis revealed that the genetic context ofant(9)-Icwas conserved inBrucella, with no mobile genetic elements found within its 20-kb surrounding region. Overall, our results demonstrate that ANT(9)-Ic is a novel member of the ANT(9)-I lineage, contributing to the intrinsic spectinomycin resistance of ZJ499.IMPORTANCEThe emergence, evolution, and worldwide spread of antibiotic resistance present a significant global public health crisis. For aminoglycoside antibiotics, enzymatic drug modification is the most common mechanism of resistance. We identify a novel chromosomal aminoglycoside nucleotidyltransferase fromB. intermedia, called ANT(9)-Ic, which shares the highest identity (47.44%) with the previously known ANT(9)-Ia and plays an important role in spectinomycin resistance of the host strain. Analysis of the genetic environment and origin ofant(9)-Icshows that the gene and its surrounding region are widely conserved inBrucella, and no mobile elements are detected, indicating that ANT(9)-Ic may be broadly important in the natural resistance to spectinomycin ofBrucellaspecies.