Tandem Multiplication of the IS26-Flanked Amplicon with the blaSHV-5 Gene within Plasmid p1658/97.

Abstract

The IncF plasmid p1658/97 (~125kb) from Escherichia coli isolates recovered during a clonal outbreak in a Warsaw, Poland, hospital 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 4μg/ml to more than 128μg/ml. Successive deletions within p1658/97, located outside the amplifiable module and encompassing even as little as ~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 favourable in p1658/97, might have been responsible for the IS26-blaSHV-5 multiplication ability.