2026-06-30T02:00:50Zhttps://repisalud.isciii.es/rest/oai/requestoai:repisalud.isciii.es:20.500.12105/82362024-09-27T20:51:04Zcom_20.500.12105_2052com_20.500.12105_2051col_20.500.12105_19609
Repisalud
author
Martin-Galiano, Antonio Javier
author
de la Campa, Adela G
funder
Comunidad de Madrid (España)
funder
Instituto de Salud Carlos III
funder
Ministerio de Ciencia y Tecnología (España)
2019-08-13T12:08:18Z
2019-08-13T12:08:18Z
2003-04
Antimicrob Agents Chemother. 2003 Apr;47(4):1257-61.
10.1128/aac.47.4.1257-1261.2003
0066-4804
Antimicrobial agents and chemotherapy
12654655
http://hdl.handle.net/20.500.12105/8236
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 beta 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 (>10(4)). This method could be used to efficiently obtain resistant mutants for any drug whose target is known.
eng
High-efficiency generation of antibiotic-resistant strains of Streptococcus pneumoniae by PCR and transformation
journal article
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HighEfficiencyGenerationOf_2003.pdf.txt
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