The objective of this study was to investigate whether the insertion sequence IS1294b (IS91 family) is able to mobilize the blaCMY-2 gene and its adjacent regions from one replicon to another. Klebsiella pneumoniae Kp2735 was typed by MLST and its plasmid content was examined by S1-PFGE and PCR-based replicon typing. The genetic blaCMY-2 environment was analysed after cloning experiments and sequencing. Transposition assays were performed with an inactivation strategy based on the sacB gene, which confers sucrose-dependent lethality. Kp2735 (ST215) exhibited high-level resistance to ceftazidime owing to the presence of the cephalosporinase CMY-2. The blaCMY-2 gene was located on an IncI1 ST156 plasmid, p2735, of ∼95 kb. Analysis of the genetic environment revealed, upstream of blaCMY-2, the presence of ISEcp1 interrupted by IS1294b and, downstream of blaCMY-2, a region of 1395 bp belonging to the backbone of IncA/C replicons, suggesting a possible DNA transfer between the two plasmids. We showed that IS1294b is able to mobilize blaCMY-2 and its adjacent regions efficiently on the recipient plasmid with a mean frequency of 5.9%. This transfer was due to a one-ended transposition mechanism, implying the non-recognition of its terIS end. Our experimental data demonstrate for the first time, to our knowledge, the mobilization of a β-lactamase gene mediated by a member of the IS91 family and highlight the important role of this mobile genetic element in the spread of antibiotic resistance genes.Read less <