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Thioridazine (TZ)-induced accumulation of the universal efflux pump substrate ethidium bromide and its subsequent efflux by Salmonella strains with various degrees of overexpressed efflux pumps takes place automatically at pH 7.4, is independent of a metabolic source, is not affected by a proton ionophore and is precluded by palmitic acid. Salmonella enterica serotype Enteritidis cultured in medium containing increasing concentrations of TZ does not grow during the first 6-8h, after which time its growth is similar to unexposed controls. At the end of a 16-h exposure period, the organism is resistant to >250mg/L TZ. Parallel assessment by real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) of the activity of genes that regulate and code for the AcrB transporter of the main efflux pump (AcrAB) of the organism at periodic intervals suggests a sequence of activation beginning with the stress gene soxS, followed by the global regulator ramA, then by the local regulator marA and then by the transporter acrB. These activations take place during the period of no growth. By the end of a 16-h culture period, only the acrB transporter gene is still highly overexpressed. Assessment of the activity of genes of the two-component regulon PmrA/B indicates that TZ also activates this regulon. Because activation of pmrA/B also activates acrB, development of high resistance to TZ during a 16-h culture period is in part due to activation of the two-component regulon., (Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

The main efflux pump of Salmonella enterica serotype Enteritidis, which obtains its energy for the extrusion of noxious agents from the proton-motive force, was studied with the aid of an ethidium bromide (EtBr) semi-automated method under conditions that define the role of metabolic energy, ions and pH in the extrusion of the universal substrate EtBr. The results obtained in this study indicate that in minimal medium containing sodium at pH 5 efflux of EtBr is independent of glucose, whereas at pH 8 metabolic energy is an absolute requirement for the maintenance of efflux. In deionised water at pH 5.5, metabolic energy is required for the maintenance of efflux. The inhibitory effect of the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) on efflux is shown to be minimised by low pH, and at high pH by metabolic energy. Similarly, thioridazine, an inhibitor of metabolic enzymes, inhibits efflux of EtBr only at pH 8 and the degree of inhibition is lessened by the presence of metabolic energy., (Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Efflux of antibiotics by Escherichia coli AG100 is performed by a variety of efflux pumps, ensuring survival of the bacterium in widely diverse media. At pH 5, efflux is independent of metabolic energy during the period of time the assay is conducted; at pH 8 it is totally dependent upon metabolic energy. Because calcium ions (Ca(2+)) are important for membrane transport channels and the activity of ATPases that provide energy functions, the role of Ca(2+) in the extrusion of an efflux pump substrate under conditions that challenge the bacterium was investigated. Real-time accumulation and efflux of ethidium bromide (EtBr) by E. coli K-12 AG100 strain [argE3 thi-1 rpsL xyl mtl Δ(gal-uvrB) supE44] was determined by a semi-automated fluorometric method in the presence and absence of Ca(2+) and agents that are known to inhibit access of calcium to enzymes that provide energy. Chlorpromazine (CPZ), an inhibitor of calcium binding to proteins (calcium-dependent enzymes), and ethylene diamine tetra-acetic acid (EDTA), a chelator of Ca(2+), increased accumulation and efflux of EtBr at pH 8 but not at pH 5. Ca(2+) reverses these effects when the assay is conducted at pH 8. In conclusion, the activity of the efflux pump system of E. coli is dependent upon metabolic energy at pH 8. Because at pH 8 hydrolysis of ATP is favoured and contributes protons for activation of the AcrAB-TolC efflux pump, CPZ is suspected of having its effects on accumulation/efflux of EtBr by indirectly affecting ATPase activity that is dependent upon Ca(2+)., (Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Six cucurbitane-type triterpenoids (1-6) isolated from the aerial parts of Momordica balsamina were evaluated for their ability to inhibit the activity of bacterial efflux pumps of methicillin-resistant Staphylococcus aureus (MRSA) COL(OXA), Enterococcus faecalis ATCC 29212, Salmonella enterica subsp. I serovar Typhimurium 5408 and S. Typhimurium 5408CIP strains. The latter strain overproduces the AcrB transporter of the AcrAB-TolC efflux pump six-fold compared with its parent. Compounds 4-6 were also tested for similar activity against Escherichia coli AG100 wild-type strain and E. coli AG100TET8 that overproduces the AcrAB-TolC efflux pump. Evaluation of efflux activity was performed using a semi-automated method that measures accumulation of the universal efflux pump substrate ethidium bromide (EtBr). Some of the compounds significantly inhibited efflux of EtBr by MRSA COL(OXA) and E. faecalis ATCC 29212. A correlation between activity and the topological polar surface area of the compounds was found for MRSA COL(OXA)., (Copyright © 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Multidrug resistance in Gram-negative bacteria is now known to be primarily caused by overexpression of efflux pumps that extrude unrelated antibiotics from the periplasm or cytoplasm of the bacterium prior to their reaching their intended target. This review focuses on a variety of agents that have been shown to be efflux pump inhibitors (EPIs) and which, if used as 'helper compounds' in combination with antibiotics to which the organism is initially resistant, may produce the required cure. Although not all of the EPIs may serve a helper role owing to their toxicity, they may nevertheless serve as lead compounds.

We previously reported that a trifluoromethyl ketone derivative, 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone (TF18), exhibited the potent antibacterial activity against Helicobacter pylori, but had no urease activity. In order to clarify the mechanism of anti-H. pylori action of TF18, we evaluated the growth and motility of TF18 on clarithromycin-susceptible H. pylori (CSHP) and -resistant H. pylori (CRHP). An effective proton pump inhibitor (TF18) had remarkable dose-dependent antibacterial activity and was able to inhibit the flagellar motor of both CSHP and CRHP isolates. The antimotility effect of TF18 was more pronounced at subinhibitory concentration in CRHP than in CSHP. The swimming (the forward motion) was more sensitive to the inhibition than the tumbling. Based on the results, it is supposed that TF18 works as an uncoupler similar to the 'clutch' in a biological motor, in which counterclockwise rotation is more sensitive to the effect of TF18 than the clockwise rotation., (Copyright 2004 Elsevier B.V.)