The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent.In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network inStaphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role thatS. aureusMDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions.

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. 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 25923_EtBr 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. Compared with its parental strain, ATCC 25923_EtBr 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 25923_EtBr could be inhibited by the same EPIs. qRT–PCR showed that norA was 35-fold over-expressed in ATCC 25923_EtBr, 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 25923_EtBr. 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.

Background: Antimicrobial resistance mediated by efflux systems is still poorly characterized in Staphylococcus aureus, despite the description of several efflux pumps (EPs) for this bacterium. In this work we used several methodologies to characterize the efflux activity of 52 S. aureus isolates resistant to ciprofloxacin collected in a hospital in Lisbon, Portugal, in order to understand the role played by these systems in the resistance to fluoroquinolones. Results: Augmented efflux activity was detected in 12 out of 52 isolates and correlated with increased resistance to fluoroquinolones. Addition of efflux inhibitors did not result in the full reversion of the fluoroquinolone resistance phenotype, yet it implied a significant decrease in the resistance levels, regardless of the type(s) of mutation(s) found in the quinolone-resistance determining region of grlA and gyrA genes, which accounted for the remaining resistance that was not efflux-mediated. Expression analysis of the genes coding for the main efflux pumps revealed increased expression only in the presence of inducing agents. Moreover, it showed that not only different substrates can trigger expression of different EP genes, but also that the same substrate can promote a variable response, according to its concentration. We also found isolates belonging to the same clonal type that showed different responses towards drug exposure, thus evidencing that highly related clinical isolates may diverge in the efflux-mediated response to noxious agents. The data gathered by real-time fluorometric and RT-qPCR assays suggest that S. aureus clinical isolates may be primed to efflux antimicrobial compounds. Conclusions: The results obtained in this work do not exclude the importance of mutations in resistance to fluoroquinolones in S. aureus, yet they underline the contribution of efflux systems for the emergence of high-level resistance. All together, the results presented in this study show the potential role played by efflux systems in the development of resistance to fluoroquinolones in clinical isolates of S. aureus.

The Rhodococcus genus contains species with remarkable ability to tolerate toxic compounds and to degrade a myriad of substrates. These substrates have to cross a distinctive cell envelope dominated by mycolic acids anchored in a scaffold of arabinogalactan covalently attached to the cell wall peptidoglycan, and a cellular membrane with phospholipids, whose composition in fatty acids can be rapidly altered in response to environmental conditions. The hydrophobic nature of the cell envelope facilitates the entrance of hydrophobic molecules but some substrates require active transport systems. Additionally, toxic compounds may also be extruded by energy spending efflux systems. In this review, physiological evidences of the use of transport systems by Rhodococcus strains and genomic studies that corroborate their existence are presented and discussed. The recently released complete genomes of several Rhodococcus strains will be the basis for an in silico correlation analysis between the efflux pumps present in the genome and their role on active transport of substrates. These transport systems will be placed on an integrative perspective of the impact of this important genus on biotechnology and health, ranging from bioremediation to antibiotic and biocide resistance.

NorA is the best studied efflux system of Staphylococcus aureus and therefore frequently used as a model for investigating efflux-mediated resistance in this pathogen. NorA activity is associated with resistance to fluoroquinolones, several antiseptics and disinfectants and several reports have pointed out the role of efflux systems, including NorA, as a first-line response to antimicrobials in S. aureus. Genetic diversity studies of the gene norA have described three alleles; norAI, norAII and norAIII. However, the epidemiology of these alleles and their impact on NorA activity remains unclear. Additionally, increasing studies do not account for norA variability when establishing relations between resistance phenotypes and norA presence or reported absence, which actually corresponds, as we now demonstrate, to different norA alleles. In the present study we assessed the variability of the norA gene present in the genome of over 1,000 S. aureus isolates, corresponding to 112 S. aureus strains with whole genome sequences publicly available; 917 MRSA strains sourced from a London-based study and nine MRSA isolates collected in a major Hospital in Lisbon, Portugal. Our analyses show that norA is part of the core genome of S. aureus. It also suggests that occurrence of norA variants reflects the population structure of this major pathogen. Overall, this work highlights the ubiquitous nature of norA in S. aureus which must be taken into account when studying the role played by this important determinant on S. aureus resistance to antimicrobials.