RESUMEN
We hypothesized that multidrug resistance efflux pumps (MDRs) may be contributing to the drug resistance of enterococci. We recently identified potential MDR-encoding genes in the Enterococcus faecalis V583 genome. Among the putative MDRs, we found a gene that encodes a NorA homolog and have characterized this enterococcal MDR in the present study. A mutant from which the enterococcal NorA homolog has been deleted has reduced resistance to several NorA substrates. Complementation of the deletion mutant with the wild-type gene verified the involvement of this enterococcal gene in resistance to ethidium bromide (EtBr) and norfloxacin. Known MDR inhibitors (reserpine, lansoprazole, and verapamil) inhibit the efflux of EtBr and norfloxacin in wild-type strain OG1RF. A fluorescence assay with EtBr allowed us to quantitate the efflux capability of the enterococcal NorA pump. On the basis of these results, we have named this enterococcal gene emeA (enterococcal multidrug resistance efflux).
Asunto(s)
Proteínas Bacterianas , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Secuencia de Aminoácidos , Cromosomas Bacterianos/genética , Medios de Cultivo , Cartilla de ADN , ADN Bacteriano/genética , Farmacorresistencia Microbiana , Resistencia a Múltiples Medicamentos , Etidio/metabolismo , Prueba de Complementación Genética , Indicadores y Reactivos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutagénesis Insercional , Plásmidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Using bioinformatics approaches, 34 potential multidrug resistance (MDR) transporter sequences representing 4 different transporter families were identified in the unannotated Enterococcus faecalis database (TIGR). A functional genomics campaign generating single-gene insertional disruptions revealed several genes whose absence confers significant hypersensitivities to known antimicrobials. We constructed specific strains, disrupted in a variety of previously unpublished, putative MDR transporter genes, as tools to improve the success of whole-cell antimicrobial screening and discovery. Each of the potential transporters was inactivated at the gene level and then phenotypically characterized, both with single disruption mutants and with 2-gene mutants built upon a delta norA deleted strain background.