Active antimicrobial efflux in Staphylococcus epidermidis: building up of resistance to fluoroquinolones and biocides in a major opportunistic pathogen

Objectives To analyse the efflux-mediated response of Staphylococcus epidermidis to ethidium bromide (EtBr), a substrate of multidrug efflux pumps (EPs). Methods The susceptible reference strain S. epidermidis ATCC 12228 was exposed to a step-wise adaptation to EtBr. The resulting EtBr-adapted strains were characterized regarding their antibiotic and biocide susceptibility by MIC determination and evaluation of efflux activity by re-determination of MICs in the presence of known efflux inhibitors and real-time fluorometry. Mutations in the QRDR of grlA and gyrA were screened by sequencing. The expression levels of S. epidermidis homologues of the main Staphylococcus aureus EP genes were quantified by RT-qPCR. Results Exposure to EtBr led to a gradual increase in resistance to antimicrobials, with the final EtBr-adapted strain, ATCC 12228_EtBr, displaying phenotypic resistance to fluoroquinolones and reduced susceptibility to several antiseptics and disinfectants, although no mutations were detected in the QRDR of the grlA/gyrA genes. A reduction in the MICs of fluoroquinolones and selected biocides promoted by efflux inhibitors suggested an efflux-mediated response to EtBr exposure. Detailed analysis of the EtBr-adapted strains detected a gradual increase in efflux activity. Gene expression assays revealed a temporal activation of S. epidermidis EPs, with an early response involving norA, SE2010 and SE1103 followed by a late response mediated by norA, which coincided with the occurrence of the mutation -1A→T in the norA promoter region. Conclusions This study demonstrated that S. epidermidis has the potential to develop a multiple resistance phenotype mediated by efflux when exposed to a non-antibiotic substrate of multidrug EPs.