Experimental evidence for IS1294b-mediated transposition of the blaCMY-2 cephalosporinase gene in Enterobacteriaceae

Objectives: The objective of this study was to investigate whether the insertion sequence IS1294b (IS91 family) is able to mobilize the bla(CMY-2) gene and its adjacent regions from one replicon to another. Methods: Klebsiella pneumoniae Kp2735 was typed by MLST and its plasmid content was examined by S1-PFGE and PCR-based replicon typing. The genetic bla(CMY-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. Results: Kp2735 (ST215) exhibited high-level resistance to ceftazidime owing to the presence of the cephalosporinase CMY-2. The bla(CMY-2) gene was located on an IncI1 ST156 plasmid, p2735, of similar to 95 kb. Analysis of the genetic environment revealed, upstream of bla(CMY-2), the presence of ISEcp1 interrupted by IS1294b and, downstream of bla(CMY-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 bla(CMY-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. Conclusions: Our experimental data demonstrate for the first time, to our knowledge, the mobilization of a beta-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.