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.

The essential oil of Helichrysum italicum significantly reduces the multidrug resistance of Enterobacter aerogenes , Escherichia coli , Pseudomonas aeruginosa , and Acinetobacter baumannii . Combinations of the two most active fractions of the essential oil with each other or with phenylalanine arginine β-naphthylamide yield synergistic activity. Geraniol, a component of one fraction, significantly increased the efficacy of β-lactams, quinolones, and chloramphenicol.

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.

Background: Efflux pump activity has been associated with multidrug resistance phenotypes in bacteria, compromising the effectiveness of antimicrobial therapy. The development of methods for the early detection and quantification of drug transport across the bacterial cell wall is a tool essential to understand and overcome this type of drug resistance mechanism. This approach was developed to study the transport of the efflux pump substrate ethidium bromide (EtBr) across the cell envelope of Escherichia coli K-12 and derivatives, differing in the expression of their efflux systems. Results: EtBr transport across the cell envelope of E. coli K-12 and derivatives was analysed by a semi-automated fluorometric method. Accumulation and efflux of EtBr was studied under limiting energy supply (absence of glucose and low temperature) and in the presence and absence of the efflux pump inhibitor, chlorpromazine. The bulk fluorescence variations were also observed by single-cell flow cytometry analysis, revealing that once inside the cells, leakage of EtBr does not occur and that efflux is mediated by active transport. The importance of AcrAB-TolC, the main efflux system of E. coli, in the extrusion of EtBr was evidenced by comparing strains with different levels of AcrAB expression. An experimental model was developed to describe the transport kinetics in the three strains. The model integrates passive entry (influx) and active efflux of EtBr, and discriminates different degrees of efflux between the studied strains that vary in the activity of their efflux systems, as evident from the calculated efflux rates: "Equation missing"<!-- image only, no MathML or LaTex --> = 0.0173 ± 0.0057 min^-1; "Equation missing"<!-- image only, no MathML or LaTex --> = 0.0106 ± 0.0033 min^-1; and "Equation missing"<!-- image only, no MathML or LaTex --> = 0.0230 ± 0.0075 min^-1. Conclusion: The combined use of a semi-automated fluorometric method and an experimental model allowed quantifying EtBr transport in E. coli strains that differ in their overall efflux activity. This methodology can be used for the early detection of differences in the drug efflux capacity in bacteria accounting for antibiotic resistance, as well as for expedite screening of new drug efflux inhibitors libraries and transport studies across the bacterial cell wall.

Membrane permeability is the first step involved in resistance of bacteria to an antibiotic. The number and activity of efflux pumps and outer membrane proteins that constitute porins play major roles in the definition of intrinsic resistance in Gram-negative bacteria that is altered under antibiotic exposure. Here we describe the genetic regulation of porins and efflux pumps of Escherichia coli during prolonged exposure to increasing concentrations of tetracycline and demonstrate, with the aid of quantitative real-time reverse transcriptase-polymerase chain reaction methodology and western blot detection, the sequence order of genetic expression of regulatory genes, their relationship to each other, and the ensuing increased activity of genes that code for transporter proteins of efflux pumps and down-regulation of porin expression. This study demonstrates that, in addition to the transcriptional regulation of genes coding for membrane proteins, the post-translational regulation of proteins involved in the permeability of Gram-negative bacteria also plays a major role in the physiological adaptation to antibiotic exposure. A model is presented that summarizes events during the physiological adaptation of E. coli to tetracycline exposure.