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Tandem multiplication of the IS26-flanked amplicon with the bla(SHV-5) gene within plasmid p1658/97
Maksymilian Zienkiewicz1, Izabela Kern-Zdanowicz, Alessandra Carattoli
1Department of Microbial Biochemistry, Institute of Biochemistry and Biophysics of Polish Academy of Sciences, Warsaw, Poland. maximus@biol.uw.edu.pl
Abstract:
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 bla(SHV-5), originated from the Klebsiella pneumoniae chromosome. A region containing the bla(SHV-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-bla(SHV-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-bla(SHV-5) multiplication ability.
Insights
The IncF plasmid p1658/97 harbors the bla(SHV-5) gene, which amplifies via IS26 elements, increasing resistance to ceftazidime. Plasmid deletions and insertions disrupt this amplification process.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Escherichia coli strains from a 1997 Warsaw hospital outbreak carried the IncF plasmid p1658/97.
- This plasmid contained the extended-spectrum β-lactamase (ESBL) gene bla(SHV-5), originating from Klebsiella pneumoniae.
Purpose of the Study:
- Investigate the mechanism of bla(SHV-5) gene amplification within the p1658/97 plasmid.
- Determine factors influencing the multiplication of IS26-bla(SHV-5) units and their impact on antibiotic resistance.
Main Methods:
- Analyzed plasmid p1658/97 structure and gene copy number.
- Studied the effect of deletions and insertions on IS26-bla(SHV-5) amplification.
- Examined the transfer of the amplifiable module to other plasmids via recombination.
Main Results:
- The bla(SHV-5) gene, flanked by IS26 copies, amplified in tandem units within p1658/97 and RecA-deficient E. coli.
- Amplification increased ceftazidime minimum inhibitory concentrations (MICs) from 4 μg/mL to over 128 μg/mL.
- Plasmid deletions outside the amplifiable module or insertions within it inhibited amplification.
Conclusions:
- Local DNA structure within p1658/97 likely facilitates IS26-bla(SHV-5) multiplication.
- The amplifiable module can transfer to other plasmids, potentially spreading antibiotic resistance.
- Understanding these mechanisms is crucial for combating the spread of ESBL-producing bacteria.
