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JAC Advance Access published online on May 28, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn217
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide

Isabel Couto1,2, Sofia Santos Costa1, Miguel Viveiros1, Marta Martins1,3 and Leonard Amaral1,3,*

1 Unidade de Micobacterias, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (UNL), Portugal 2 Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Portugal 3 UPMM, Instituto de Higiene e Medicina Tropical, UNL, Portugal

Received 21 January 2008; returned 27 February 2008; revised 17 April 2008; accepted 21 April 2008

* Correspondence address. Unidade de Micobacterias, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisbon, Portugal. Tel: +351-21-3652652; Fax: +351-21-3632105; E-mail: lamaral{at}ihmt.unl.pt

Objectives: By adapting an antibiotic-susceptible Staphylococcus aureus strain to increasing concentrations of ethidium bromide, a known substrate of efflux pumps (EPs), and by phenotypically and genotypically analysing the resulting progeny, we characterized the molecular mechanisms of S. aureus adaptation to ethidium bromide.

Methods: S. aureus ATCC 25923 was grown in increasing concentrations of ethidium bromide. The MICs of representatives of eight classes of antibiotics, eight biocides and two dyes against ATCC 25923 and its ethidium bromide-resistant progeny ATCC 25923EtBr were determined with or without six efflux pump inhibitors (EPIs). Efflux activity in the presence/absence of EPIs was evaluated by real-time fluorometry. The presence and expression of eight EP genes were assayed by PCR and quantitative RT–PCR (qRT–PCR), respectively. Mutations in grlA, gyrA and norA promoter regions were screened by DNA sequencing.

Results: Compared with its parental strain, ATCC 25923EtBr was 32-fold more resistant to ethidium bromide and also more resistant to biocides and hydrophilic fluoroquinolones. Resistance to these could be reduced by the EPIs chlorpromazine, thioridazine and reserpine. Increased efflux of ethidium bromide by ATCC 25923EtBr could be inhibited by the same EPIs. qRT–PCR showed that norA was 35-fold over-expressed in ATCC 25923EtBr, whereas the remaining EP genes showed no significant increase in their expression. Sequencing of the norA promoter region revealed a 70 bp deletion in ATCC 25923EtBr.

Conclusions: Exposure of S. aureus to quaternary compounds such as ethidium bromide results in decreased susceptibility of the organism to a wide variety of compounds, including quinolones and biocides through an efflux-mediated response, which for strain ATCC 25923 is mainly NorA-mediated. This altered expression may result from alterations in the norA promoter region.

Key Words: staphylococci , efflux pumps , NorA , overexpression


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