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dc.contributor.authorMartin-Galiano, Antonio Javier 
dc.contributor.authorde la Campa, Adela G
dc.identifier.citationAntimicrob Agents Chemother. 2003 Apr;47(4):1257-61.es_ES
dc.description.abstractWe 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.es_ES
dc.description.sponsorshipWe thank E. García for critical reading of the manuscript. The technical assistance of Amaya Aguirre is acknowledged. A.J.M.-G. is the recipient of a fellowship from the Comunidad Autónoma de Madrid. This study was supported by grant 1274/01 from the Instituto de Salud Carlos III, grant 00/0258 from the Fondo de Investigación Sanitaria, and grant BIO2002-01398 from the Ministerio de Ciencia y Tecnología.es_ES
dc.publisherAmerican Society for Microbiologyes_ES
dc.subject.meshAmino Acid Sequence es_ES
dc.subject.meshCiprofloxacin es_ES
dc.subject.meshMolecular Sequence Data es_ES
dc.subject.meshPolymerase Chain Reaction es_ES
dc.subject.meshRifampin es_ES
dc.subject.meshStreptococcus pneumoniae es_ES
dc.subject.meshStreptomycin es_ES
dc.subject.meshDrug Resistance, Bacterial es_ES
dc.subject.meshTransformation, Bacterial es_ES
dc.titleHigh-efficiency generation of antibiotic-resistant strains of Streptococcus pneumoniae by PCR and transformationes_ES
dc.rights.licenseAtribución-NoComercial-CompartirIgual 4.0 Internacional*
dc.contributor.funderComunidad de Madrid
dc.contributor.funderFondo de Investigaciones Sanitarias
dc.contributor.funderInstituto de Salud Carlos III - ISCIII
dc.contributor.funderMinisterio de Ciencia y Tecnología (España)
dc.identifier.journalAntimicrobial agents and chemotherapyes_ES
dc.repisalud.centroISCIII::Centro Nacional de Microbiologíaes_ES

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