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Antimicrobial Agents and Chemotherapy, April 2003, p. 1257-1261, Vol. 47, No. 4
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.4.1257-1261.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

High-Efficiency Generation of Antibiotic-Resistant Strains of Streptococcus pneumoniae by PCR and Transformation

Unidad de Genética Bacteriana (Consejo Superior de Investigaciones Científicas), Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain

Received 8 November 2002/ Returned for modification 2 January 2003/ Accepted 21 January 2003

We designed a method by which to generate antibiotic-resistant strains of Streptococcus pneumoniae at frequencies 4 orders of magnitude greater than the spontaneous mutation rate. The method is based on the natural ability of this organism to be genetically transformed with PCR products carrying sequences homologous to its chromosome. The genes encoding the targets of ciprofloxacin (parC, encoding the ParC subunit of DNA topoisomerase IV), rifampin (rpoB, encoding the ß subunit of RNA polymerase), and streptomycin (rpsL, encoding the S12 ribosomal protein) from susceptible laboratory strain R6 were amplified by PCR and used to transform the same strain. Resistant mutants were obtained with a frequency of 10^-4 to 10^-5, depending on the fidelity of the DNA polymerase used for PCR amplifications. Ciprofloxacin-resistant mutants, for which the MICs were four-to eightfold higher than that for R6, carried a single mutation of a residue in the quinolone resistance-determining region: S79 (change to A, F, or Y) or D83 (change to N or V). Rifampin-resistant strains, for which the MICs were at least 133-fold higher than that for R6, contained a single mutation within cluster I of rpoB: S482 (change to P), Q486 (change to L), D489 (change to V), or H499 (change to L or Y). Streptomycin-resistant mutants, for which the MICs were at least 64-fold higher than that for R6, carried a mutation at either K56 (change to I, R, or T) or K101 (change to E). PCR products obtained from the mutants were able to transform R6 to resistance with high efficiency (>10⁴). This method could be used to efficiently obtain resistant mutants for any drug whose target is known.

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