High levels of resistance to aminoglycosides conferred by plasmid-mediated mechanisms of resistance to aminoglycosides corresponding to 16S rRNA methylases have been reported since 2003 (3, 11). To date, six enzymes (ArmA, RmtA, RmtB, RmtC, RmtD, and NpmA) have been described, with ArmA being the most frequently identified methylase in Enterobacteriaceae (2). Associations between 16S rRNA methylase- and extended-spectrum -lactamase (ESBL)-encoding genes such as the blaCTX-M genes have been reported (1, 7, 8). The aim of this study was to evaluate the prevalence of 16S rRNA methylase genes among ESBL-producing Enterobacteriaceae isolates recovered in a French university hospital. A total of 373 nonduplicate ESBL-producing enterobacterial clinical isolates, collected at the Bicêtre hospital between January 2005 and December 2007, have been studied. Among them, 19 isolates (5.1%) were resistant to all clinically used aminoglycosides. That collection was screened by PCR for detection of 16S rRNA methylase-encoding genes (armA, rmtA, rmtB, rmtC, rmtD, and npmA), as previously described (2). Four armA-positive isolates were detected in 2005 (n 2) and in 2007 (n 2), in addition to a single rmtB-positive isolate from 2006. No isolate was positive for the rmtA, rmtC, rmtD, and npmA genes. The armA-positive isolates produced the ESBL CTX-M-3 (n 3) or CTX-M-14 (n 1), whereas the single rmtB-positive isolate was a CTX-M-14 producer. The low prevalence rate of 16S rRNA methylase determinants among ESBL producers (1.3%) observed here is similar to that reported from Taiwan (1.1%) (8) and higher than those reported from Belgium (0.12%) and Japan (0.03%) (1, 10), but lower than that from Korea (3%) (7). All 16S rRNA methylase and ESBL determinants were transferred by conjugation and transformation using Escherichia coli J53 and TOP10 recipient strains after selection on Trypticase soy agar containing amikacin (50 g/ml) and/or cefotaxime (8 g/ml) (6). The 16S rRNA methylase-positive transformants expressed a high level of resistance to aminoglycosides (Table 1). In three isolates, the armA and blaCTX-M-3 genes were located on a same plasmid belonging to the IncL/M incompatibility group, as observed on the plasmid pCTX-M-3 (5), whereas the 16S rRNA methylase determinants were not physically linked to the ESBL genes for the two blaCTX-M-14 positive remaining isolates. Sequencing of the 16S rRNA methylase-encoding genes showed a perfect identity with previously reported genes. The complete PCR-mapped region of 11.6 kb containing the armA gene was entirely sequenced for two isolates, showing its association with ISCR1 inside a sul1 type integron structure, the same configuration observed in Tn1548 (4). The rmtB gene was located on an 11.4-kb region bracketed by two IS26 elements containing tnpA, blaTEM-1, intI1/groEL, and qepA and followed by an ISCR3 element, as observed on pHPA (9). Analysis of sequences flanking more closely the armA gene identified two novel insertion sequence elements, ISEc28 and ISEc29, belonging to the IS5 and IS4 families, respectively (http://www-is.biotoul.fr). No direct repeat sequence bracketing each of those IS elements and the overall ISEc28-armA-ISEc29 structure was identified. This study underlines that the association on the same plasmid of 16S rRNA methylase- and ESBL-encoding genes remains rare. However, their localization on conjugative plasmids, and in some cases in association on the same plasmid, as observed here with CTX-M determinants, has the potential to further expand the threat of multidrug-resistant Enterobacteriaceae.