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Antimicrobial Agents and Chemotherapy, February 2003, p. 682-688, Vol. 47, No. 2
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.2.682-688.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Tandem Amplification of a 28-Kilobase Region from the Yersinia enterocolitica Chromosome Containing the blaA Gene

Departamento de Biología Molecular, Unidad Asociada al CIB, CSIC, Facultad de Medicina, Universidad de Cantabria, Cardenal Herrera Oria s/n, 3901-Santander, Spain

Received 9 August 2002/ Returned for modification 15 October 2002/ Accepted 14 November 2002

Most Yersinia enterocolitica strains are resistant to ß-lactam antibiotics due to the production of one or two chromosomally encoded ß-lactamases. Strain Y56 is a Y. enterocolitica O:3 serotype natural isolate that is resistant to moderate amounts of penicillins and that produces a single class A ß-lactamase. To select mutants with increased levels of resistance to ß-lactam antibiotics, strain Y56 was grown on plates containing increasing amounts of ampicillin, and variants resistant to up to 500 µg of ampicillin per ml were obtained. Chromosomal DNA from hyperresistant isolates was analyzed by Southern hybridization with a blaA-specific probe to detect gene rearrangements. The use of pulsed-field gel electrophoresis revealed that the increase in the resistance level correlated with the amplification in tandem of a DNA fragment of about 28 kb containing the blaA gene. The phenotype of these isolates was not stable, and they recovered the basal low resistance level when the ampicillin used for selection was withdrawn from the growth medium. This loss of resistance was followed by the recovery of the original chromosomal structure. To understand this amplification process, the 28-kb amplification unit was cloned, and the ends were sequenced. The analysis of these sequences did not reveal the presence of either repeats or transposable elements to explain this process. However, we found short sequences similar to some DNA gyrase target sequences that have been described. In addition, we observed that the frequency of appearance of ampicillin-hyperresistant isolates by amplification of the blaA locus was lowered in the presence of the gyrase inhibitor novobiocin. These findings suggest that the DNA gyrase could be involved in this amplification event.

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