Complete nucleotide sequence of the large conjugative pTC2 multireplicon plasmid encoding the VIM-1 metallo-β-lactamase.

To determine the complete nucleotide sequence of the VIM-1-encoding plasmid pTC2, which was isolated from a Greek Providencia stuartii multiresistant strain. The pTC2 plasmid was extracted and sequenced using shotgun and 3 kb paired-end DNA libraries and a 454 sequencing approach. Following assembly into a unique scaffold, gaps were closed by PCR followed by Sanger DNA sequencing. Gene predictions and annotations were performed using the CAAT-Box tool and the Conserved Domain Search service. Sequence comparisons were performed using the Artemis Comparison Tool. Plasmid pTC2 (180184 bp) was found to be a multireplicon plasmid (IncA/C and IncR), with a large IncA/C backbone and a mosaic multidrug resistance (MDR) region, in which was inserted a 13 kb IncR fragment. Gene annotation allowed the identification of a complete IncA/C-type transfer system and of several putative maintenance modules, both on the IncA/C backbone and on the IncR fragment. The complex MDR region contained 9 insertion sequences (7 IS26, 1 IS1 and 1 IS6100), 10 resistance genes and a mercury resistance operon integrated into unit transposons, composite transposons or integrons. The pTC2 combines a broad host range, transfer and maintenance capacities, plasticity of the MDR region and a wide variety of resistance genes, properties that may contribute to the spreading of resistance determinants.