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

Abstract

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 themain Staphylococcus aureus EP geneswere 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.