c 16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. Aminoglycosides play an important role in treating serious infections caused by Gram-positive and -negative organisms, with amikacin, gentamicin, and tobramycin being of particular clinical value (1). In Gram-negative organisms, resistance is primarily mediated by aminoglycoside-modifying enzymes (AMEs), which inactivate the antibiotic through modification of the -OH or -NH 2 groups (2). AMEs are divided into three classes: the ATP (and/or GDP)-dependent aminoglycoside phosphotransferases (APHs), the acetyl-coenzyme A (CoA)-dependent aminoglycoside acetyltransferases (AACs), and the ATP-dependent aminoglycoside nucleotidyltransferases (ANTs) (2). Different classes have different antibiotic substrates, but no single AME confers resistance to all aminoglycosides. In the past decade, transmissible 16S rRNA methyltransferases (16S RMTases) have emerged as a new and worrisome source of aminoglycoside resistance that operates by reducing the affinity of nearly all aminoglycosides for 16S rRNA via ribosomal methylation (3). Since the presence of multiple AMEs can confer resistance to all clinically relevant aminoglycosides, discriminating between AME RMTase activity and 16S RMTase activity using phenotypic methods can be challenging (3).Arbekacin is a semisynthetic aminoglycoside derived from dibekacin (4). It is licensed only in Japan, where it is used to treat septicemia and pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA). However, it also has broad antibacterial activity against Gram-negative bacteria, including strains that are resistant to amikacin, gentamicin, and tobramycin (5). Recently, the U.S. Food and Drug Administration approved arbekacin for use under a protocol at the Walter Reed National Military Medical Center to treat infections for which no other antibiotic is available. Importantly, arbekacin is refractory to nearly all AMEs with the exception of some members of the AAC(6=)-I family and the bifunctional enzyme AAC(6=)/APH(2==) produced by some strains of S. aureus and enterococci (3,4,(6)(7)(8). However, even though these enzymes acetylate arbekacin, the antibiotic still retains some antimicrobial activity (9). We exploited this characteristic to develop a rapid method to detect 16S RMTase activity in Enterobacteriaceae using arbekacin-impregnated susceptibility disks.125 Escherichia coli, 74 Klebsiella pneumoniae, and 22 other Enterobacteriaceae isolates, including Enterobacter (n ϭ 4), Morganella (n ϭ 2), Proteus (n ϭ 3), Providencia (n ϭ 6), Serratia (n ϭ