Tandem multiplication of the IS26-flanked amplicon with the blaSHV-5 gene within plasmid p1658/97

The IncF plasmid p1658/97 (c. 125 kb) from Escherichia coli isolates recovered during a clonal outbreak in a hospital in Warsaw, Poland, in 1997 contains the extended-spectrum β-lactamase (ESBL) gene blaSHV-5, originated from the Klebsiella pneumoniae chromosome. A region containing the blaSHV-5 gene is flanked by two IS26 copies and its copy number multiplies spontaneously within p1658/97 and RecA-deficient E. coli strains. Here, we demonstrate that the amplified IS26-blaSHV-5 units were arranged in tandems, containing up to more than 10 units, which could raise ceftazidime MICs for host strains from 4 μg mL-1 to more than 128 μg mL-1. Successive deletions within p1658/97, located outside the amplifiable module and encompassing even as little as c. 15% of the plasmid, blocked the amplification. Moreover, the complementing re-introduction of the deleted fragments in trans did not restore the process. Similarly, insertions of a 1-kb DNA fragment into the amplicon inhibited its self-multiplication ability. The module was able to transmit into another IS26-containing plasmid by recombination. The results prompted us to speculate that local DNA structure, especially favorable in p1658/97, might have been responsible for the IS26-blaSHV-5 multiplication ability. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.