Characterization of Amino Acid Substitutions in the Two-Component Regulatory System AdeRS Identified in Multidrug-Resistant Acinetobacter baumannii.

In Acinetobacter baumannii, resistance-nodulation-cell division (RND)-type efflux is a resistance mechanism of great importance since it contributes to reduced susceptibility to multiple antimicrobial compounds. Some mutations within the genes encoding the two-component regulatory system AdeRS appear to play a major role in increased expression of the RND efflux pump AdeABC and, consequently, in reduced antimicrobial susceptibility, as they are commonly observed in multidrug-resistant (MDR) A. baumannii. In the present study, the impact of frequently identified amino acid substitutions, namely, D21V and D26N in AdeR and T156M in AdeS, on adeB expression, efflux activity, and antimicrobial susceptibility was investigated. Reverse transcription-quantitative PCR (qRT-PCR) studies revealed significantly increased adeB expression caused by D26N (AdeR) and T156M (AdeS). In addition, accumulation assays have shown that these mutations induce increased efflux activity. Subsequently, antimicrobial susceptibility testing via agar dilution and broth microdilution confirmed the importance of these substitutions for the MDR phenotype, as the MICs for various antimicrobials of different classes were increased. In contrast, the amino acid substitution D21V in AdeR did not lead to increased adeB expression and did not reduce antimicrobial susceptibility. This study demonstrates the impact of the D26N (AdeR) and T156M (AdeS) amino acid substitutions, highlighting that these regulators represent promising targets for interfering with efflux activity to restore antimicrobial susceptibility. IMPORTANCE The active efflux of antimicrobials by bacteria can lead to antimicrobial resistance and persistence and can affect multiple different classes of antimicrobials. Efflux pumps are tightly regulated, and their overexpression can be mediated by changes in their regulators. Identifying these changes is one step in the direction of resistance prediction, but it also opens the possibility of targeting efflux pump regulation as a strategy to overcome antimicrobial resistance. Here, we have investigated commonly found changes in the regulators of the main efflux pumps in Acinetobacter baumannii.

In silico and in vitro evaluation of tetrahydropyridine compounds as efflux inhibitors in Mycobacterium abscessus.

Herein, we evaluated tetrahydropyridine (THP) compounds (NUNM) as antimicrobials and inhibitors of the efflux mechanism in M. abscessus. subsp. abscessus. The modulation factor (MF) of efflux inhibitors was calculated from the minimum inhibitory concentrations (MICs) of amikacin (AMI), ciprofloxacin (CIP) and clarithromycin (CLA) in the absence and presence of subinhibitory concentrations of the NUNM compounds and canonical inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and verapamil (VP). The kinetics of the intracellular accumulation of the fluorimetric substrate ethidium bromide (EtBr) was evaluated and calculated by the relative final fluorescence (RFF). In addition, molecular modeling simulations for the MmpL5 and Tap efflux transporters with ligands (CLA, NUNM, CCCP, VP and EtBr) were performed to better understand the efflux mechanism. We highlight the NUNM01 compound because it reduced the MICs of AMI, CIP and CLA by 4-, 4- and 16-fold, respectively, had the highest effect on EtBr accumulation (RFF = 3.1) and showed a significant in silico affinity for the evaluated proteins in docking simulations. Based on the analyses performed in vitro and in silico, we propose that NUNM01 is a potential pharmacophore candidate for the development of a therapeutic adjuvant for M. abscessus infections.

Powering the ABC multidrug exporter LmrA: How nucleotides embrace the ion-motive force.

LmrA is a bacterial ATP-binding cassette (ABC) multidrug exporter that uses metabolic energy to transport ions, cytotoxic drugs, and lipids. Voltage clamping in a Port-a-Patch was used to monitor electrical currents associated with the transport of monovalent cationic HEPES+ by single-LmrA transporters and ensembles of transporters. In these experiments, one proton and one chloride ion are effluxed together with each HEPES+ ion out of the inner compartment, whereas two sodium ions are transported into this compartment. Consequently, the sodium-motive force (interior negative and low) can drive this electrogenic ion exchange mechanism in cells under physiological conditions. The same mechanism is also relevant for the efflux of monovalent cationic ethidium, a typical multidrug transporter substrate. Studies in the presence of Mg-ATP (adenosine 5'-triphosphate) show that ion-coupled HEPES+ transport is associated with ATP-bound LmrA, whereas ion-coupled ethidium transport requires ATP binding and hydrolysis. HEPES+ is highly soluble in a water-based environment, whereas ethidium has a strong preference for residence in the water-repelling plasma membrane. We conclude that the mechanism of the ABC transporter LmrA is fundamentally related to that of an ion antiporter that uses extra steps (ATP binding and hydrolysis) to retrieve and transport membrane-soluble substrates from the phospholipid bilayer.

The Asp20-to-Asn Substitution in the Response Regulator AdeR Leads to Enhanced Efflux Activity of AdeB in Acinetobacter baumannii.

Overexpression of the resistance-nodulation-cell division-type efflux pump AdeABC is often associated with multidrug resistance in Acinetobacter baumannii and has been linked to mutations in the genes encoding the AdeRS two-component system. In a previous study, we reported that the Asp20→Asn amino acid substitution in the response regulator AdeR is associated with adeB overexpression and reduced susceptibility to the antimicrobials levofloxacin, tigecycline, and trimethoprim-sulfamethoxazole. To further characterize the effect of the Asp20→Asn substitution on antimicrobial susceptibility, the expression of the efflux genes adeB, adeJ, and adeG, and substrate accumulation, four plasmid constructs [containing adeR(Asp20)S, adeR(Asn20)S, adeR(Asp20)SABC, and adeR(Asn20)SABC] were introduced into the adeRSABC-deficient A. baumannii isolate NIPH 60. Neither adeRS construct induced changes in antimicrobial susceptibility or substrate accumulation from that for the vector-only control. The adeR(Asp20)SABC transformant showed reduced susceptibility to 6 antimicrobials and accumulated 12% less ethidium than the control, whereas the Asn20 variant showed reduced susceptibility to 6 of 8 antimicrobial classes tested, and its ethidium accumulation was only 72% of that observed for the vector-only construct. adeB expression was 7-fold higher in the adeR(Asn20)SABC transformant than in its Asp20 variant. No changes in adeG or adeJ expression or in acriflavine or rhodamine 6G accumulation were detected. The antimicrobial susceptibility data suggest that AdeRS does not regulate any resistance determinants other than AdeABC. Furthermore, the characterization of the Asp20→Asn20 substitution proves that the reduced antimicrobial susceptibility previously associated with this substitution was indeed caused by enhanced efflux activity of AdeB.

Influence of induced ciprofloxacin resistance on efflux pump activity of Klebsiella pneumoniae.

OBJECTIVE: The efflux pump (EP) is one of the major mechanisms of antibiotic resistance in Klebsiella pneumoniae. However, there are few reports on the effect of the abuse of antibiotic use on the activity of EPs. To determine whether the use of low efficacy antibiotics has any effect on the activity of EPs and induces drug resistance in K. pneumoniae, we investigated the effect of ciprofloxacin on the activity of EPs in K. pneumoniae strains. METHODS: Sixteen susceptible K. pneumoniae strains were isolated from patients and their minimum inhibitory concentrations (MICs) of ciprofloxacin were measured in the absence and presence of the pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The strains were then induced with a gradient of ciprofloxacin until the MICs of the strains showed no further increase, to obtain induced resistant strains. The EP activities of the strains before and after induction were compared using EP inhibition and ethidium bromide (EtBr) accumulation assays. RESULTS: The MIC values of the strains were 16‒256 times higher after induction than before induction. In the presence of CCCP, the MIC values of 50% of the induced strains were 2‒4-fold lower than that in the absence of this inhibitor. The EtBr accumulation assay showed that the fluorescence of EtBr in the induced cells was lower than that in the cells before induction. CONCLUSIONS: EPs are widespread in susceptible and drug-resistant K. pneumoniae strains. Induction with ciprofloxacin may increase the activity of EPs in K. pneumoniae. The EtBr accumulation assay is more sensitive than the EP inhibition assay in evaluating the activity of EPs in K. pneumoniae.

Carnosic acid is an efflux pumps modulator by dissipation of the membrane potential in Enterococcus faecalis and Staphylococcus aureus.

Bacterial resistance to antibiotics has become a serious problem of public health. Along with the controlled permeability by the cell-wall, active efflux systems can provide resistance by extruding antibiotics. Carnosic acid is capable to potentiate the antimicrobial activity of several antibiotics. However, the underlying molecular mechanism governing this effect remains unclear. The present study aims to investigate the effect of carnosic acid on the transport of ethidium bromide, on the permeability or the membrane potential in Enterococcus faecalis and Staphylococcus aureus. By using fluorimetric assays it was demonstrated that in E. faecalis, carnosic acid is a modulator of the uptake and efflux of ethidium bromide which does not induce cell membrane permeabilization phenomena. Such effect was sensitive to the inhibition caused by both the proton-motive force carbonyl cyanide m-chlorophenylhydrazone and the calcium antagonist verapamil, but not to vanadate, an ATPase inhibitor. In this work it was demonstrated, for the first time, that the activity of carnosic acid on the uptake/efflux of ethidium bromide is correlated with its capacity to change the membrane potential gradient in S. aureus and E. faecalis. In conclusion, carnosic acid is a natural compound, structurally unrelated to known antibiotics, which can function as an efflux pump modulator by dissipation of the membrane potential. Therefore, carnosic acid would be a good candidate to be employed as a novel therapeutic agent to be used in combination therapies against drug-resistant enterococci and S. aureus infections.

Splice variants of the P2X7 receptor reveal differential agonist dependence and functional coupling with pannexin-1.

P2X7 receptors function as ATP-gated cation channels but also interact with other proteins as part of a larger signalling complex to mediate a variety of downstream responses that are dependent upon the cell type in which they are expressed. Receptor-mediated membrane permeabilization to large molecules precedes the induction of cell death, but remains poorly understood. The mechanisms that underlie differential sensitivity to NAD are also unknown. By studying alternative variants of the mouse P2X7 receptor we show that sensitivity to NAD is mediated through the P2X7k variant, which has a much more restricted distribution than the P2X7a receptor, but is expressed in T lymphocytes. The altered N-terminus and TM1 of the P2X7k receptor enhances the stability of the active state of this variant compared with P2X7a, thereby increasing the efficacy of NAD-dependent ADP ribosylation as measured by ethidium uptake, a rise in intracellular Ca(2+) and the activation of inward currents. Co-expression of P2X7k and P2X7a receptors reduced NAD sensitivity. P2X7k-receptor-mediated ethidium uptake was also triggered by much lower BzATP concentrations and was insensitive to the P451L single nucleotide polymorphism. P2X7k-receptor-mediated ethidium uptake occurred independently of pannexin-1 suggesting a pathway intrinsic to the receptor. Only for the P2X7aL451 receptor could we resolve a component of dye uptake dependent upon pannexin-1. Signalling occurred downstream of the activation of caspases rather than involving direct cross talk between the channels. However, an in situ proximity assay showed close association between P2X7 receptors and pannexin-1, which would facilitate ATP efflux through pannexin-1 acting in an autocrine manner.

Dynamic optical imaging of metabolic and NADPH oxidase-derived superoxide in live mouse brain using fluorescence lifetime unmixing.

Superoxide is the single-electron reduction product of molecular oxygen generated by mitochondria and the innate immune enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), and its isoforms. Initially identified as critical to the host defense against infection, superoxide has recently emerged as an important signaling molecule and as a proposed mediator of central nervous system injury in stroke, neurodegenerative conditions, and aging itself. Complete understanding of superoxide in central nervous system disease has been hampered by lack of noninvasive imaging techniques to evaluate this highly reactive, short-lived molecule in vivo. Here we describe a novel optical imaging technique to monitor superoxide real time in intact animals using a fluorescent probe compound and fluorescence lifetime contrast-based unmixing. Specificity for superoxide was confirmed using validated mouse models with enhanced or attenuated brain superoxide production. Application of fluorescence lifetime unmixing removed autofluorescence, further enhanced sensitivity and specificity of the technique, permitted visualization of physiologically relevant levels of superoxide, and allowed superoxide in specific brain regions (e.g., hippocampus) to be mapped. Lifetime contrast-based unmixing permitted disease model-specific and brain region-specific differences in superoxide levels to be observed, suggesting this approach may provide valuable information on the role of mitochondrial and Nox-derived superoxide in both normal function and pathologic conditions in the central nervous system.

Identification of efflux pump-mediated multidrug-resistant bacteria by the ethidium bromide-agar cartwheel method.

BACKGROUND/AIM: Bacterial multidrug resistance may be mediated by the overexpression of efflux pumps. Conventional evaluation of efflux activity using efflux pump substrates, such as ethidium bromide, requires specialised instrumentation. The agar-based method, previously reported, has been modified to evaluate as many as twelve bacterial strains and has been termed the ethidium bromide-agar cartwheel method. MATERIALS AND METHODS: Agar plates containing different concentrations of ethidium bromide were swabbed with bacterial cultures. The cell efflux capacity increased with increasing ethidium bromide concentration, which produced fluorescence of the bacterial mass. RESULTS: The method was shown to be useful for the detection of efflux activity among multidrug-resistant Gram-negative and Gram-positive clinical isolates, as confirmed by the determination of minimum inhibitory concentration for several antibiotics in the presence of known efflux pump inhibitors. CONCLUSION: This method may be adapted to the clinical laboratory for the presumptive identification of multidrug-resistant isolates that overexpress efflux pump systems.

Goldenseal (Hydrastis canadensis L.) extracts synergistically enhance the antibacterial activity of berberine via efflux pump inhibition.

Goldenseal (Hydrastis canadensis L.) is used to combat inflammation and infection. Its antibacterial activity in vitRO has been attributed to its alkaloids, the most abundant of which is berberine. The goal of these studies was to compare the composition, antibacterial activity, and efflux pump inhibitory activity of ethanolic extracts prepared from roots and aerial portions of H. canadensis. Ethanolic extracts were prepared separately from roots and aerial portions of six H. canadensis plants. Extracts were analyzed for alkaloid concentration using LC-MS and tested for antimicrobial activity against Staphylococcus aureus (NCTC 8325-4) and for inhibition of ethidium bromide efflux. Synergistic antibacterial activity was observed between the aerial extract (FIC 0.375) and to a lesser extent the root extract (FIC 0.750) and berberine. The aerial extract inhibited ethidium bromide efflux from wild-type S. aureus but had no effect on the expulsion of this compound from an isogenic derivative deleted for norA. Our studies indicate that the roots of H. canadensis contain higher levels of alkaloids than the aerial portions, but the aerial portions synergize with berberine more significantly than the roots. Furthermore, extracts from the aerial portions of H. canadensis contain efflux pump inhibitors, while efflux pump inhibitory activity was not observed for the root extract. The three most abundant H. canadensis alkaloids, berberine, hydrastine, and canadine, are not responsible for the efflux pump inhibitory activity of the extracts from H. canadensis aerial portions.

Biological activity of hydantoin derivatives on P-glycoprotein (ABCB1) of mouse lymphoma cells.

BACKGROUND: Hydantoin derivatives possess a variety of biochemical and pharmacological properties. Although hydantoin compounds are studied extensively, there are not many studies that investigate their anticancer properties. MATERIALS AND METHODS: Thirty hydantoin compounds were evaluated for their efflux modulating effects in cancer cells using a rhodamine 123 accumulation assay and real-time fluorometry based on the intracellular accumulation of ethidium bromide. RESULTS: The 30 derivatives were screened by real-time fluorometry for rhodamine 123 accumulation. Among the selected derivatives, compounds SZ-7, LL-9, BS-1, MN-3, P3, RW-15b, AD-26, RW-13, AD-29 and KF-2 significantly increased the retention of rhodamine 123. Compounds AD-26, AD-29, RW-13, KF-2, BS-1, MN-3, RW-15b and JH-63 showed synergistic effect with doxorubicin on mouse lymphoma cells. Furthermore, compound SZ-7 had indifferent effect with doxorubicin. CONCLUSION: These results indicated the role of chemical modifications within the hydantoin ring for its potential inhibition of the ABCB1 transporter. The most active structures contained aromatic substituents as well as some tertiary amine fragments.

Two haplotypes of the P2X(7) receptor containing the Ala-348 to Thr polymorphism exhibit a gain-of-function effect and enhanced interleukin-1beta secretion.

The P2X(7) receptor is an ATP-gated cation channel expressed in immune cells and plays a role in proinflammatory cytokine release from monocytes and macrophages. This study investigated the coinheritance of 12 functionally relevant single nucleotide polymorphisms (SNPs) in the human P2X(7) gene (P2RX7), and the functional effect of each singly and in combination was assessed by measurements of ATP-induced currents and ethidium(+) uptake. Genotyping of 3430 Caucasian subjects identified 4 common haplotypes in addition to the common (wild-type) P2X(7)-1. Two haplotypes (denoted P2X(7)-2 and P2X(7)-4) contained various combinations of gain-of-function SNPs. P2X(7)-4 was identified uniquely by the Gln-460 to Arg polymorphism (rs2230912). When expressed in HEK-293 cells, recombinant P2X(7)-2, and P2X(7)-4 haplotypes displayed a 3-fold and 5-fold increase, respectively, in receptor function compared to the wild-type P2X(7)-1. Both P2X(7) haplotypes contained the Ala-348>Thr polymorphism (rs1718119), and this mutation was critical for the gain-of-function effect. Peripheral blood monocytes and erythrocytes from subjects homozygous for gain-of-function P2X(7) haplotypes exhibited increased ATP-induced ethidium(+) uptake and (86)Rb(+) efflux, respectively, and this correlated with increased IL-1beta secretion from LPS-primed monocytes. Inheritance of these P2X(7) haplotypes predisposing to increased proinflammatory cytokine secretion may be important in genetic association studies of inflammatory, infectious, and psychiatric disorders.

Organic cation transporters OCT1, 2, and 3 mediate high-affinity transport of the mutagenic vital dye ethidium in the kidney proximal tubule.

The positively charged fluorescent dyes ethidium (Et(+)) and propidium (Pr(2+)) are widely used as DNA and necrosis markers. Et(+) is cytotoxic and mutagenic. The polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2), and OCT3 (SLC22A3) mediate electrogenic facilitated diffusion of small (< or =500 Da) organic cations with broad specificities. In humans, OCT2 mediates basolateral uptake by kidney proximal tubules (PT), whereas in rodents OCT1/2 are involved. In mouse kidney, perfused Et(+) accumulated predominantly in the S2/S3 segments of the PT, but not Pr(2+). In cells stably overexpressing human OCTs (hOCTs), Et(+) uptake was observed with K(m) values of 0.8 +/- 0.2 microM (hOCT1), 1.7 +/- 0.5 microM (hOCT2), and 2.0 +/- 0.5 microM (hOCT3), whereas Pr(2+) was not transported. Accumulation of Et(+) was inhibited by OCT substrates quinine, 3-methyl-4-phenylpyridinium (MPP(+)), cimetidine, and tetraethylammonium (TEA(+)). For hOCT1 and hOCT2, the IC(50) values for MPP(+), TEA(+), and cimetidine were higher than for inhibition of previously tested transported substrates. For hOCT2, the inhibition of Et(+) uptake by MPP(+) and cimetidine was shown to be competitive. Et(+) also inhibited transport of 0.1 microM [(3)H]MPP(+) by all hOCT isoforms with IC(50) values between 0.4 and 1.3 microM, and the inhibition of hOCT1-mediated uptake of MPP(+) by Et(+) was competitive. In Oct1/2(-/-) mice, Et(+) uptake in the PT was almost abolished. The data demonstrate that Et(+) is taken up avidly by the PT, which is mediated by OCT1 and/or OCT2. Considering the high affinity of OCTs for Et(+) and their strong expression in various organs, strict safety guidelines for Et(+) handling should be reinforced.

Synthesis and structure-activity relationships of novel, substituted 5,6-dihydrodibenzo[a,g]quinolizinium P2X7 antagonists.

Iminium quaternary protoberberine alkaloids (QPA) have been found to be novel P2X(7) antagonists. To assess their structure-activity relationships, these compounds were modified at their R(1) and R(2) groups and assayed for their ability to inhibit the 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP)-induced uptake of fluorescent ethidium by HEK-293 cells stably expressing the human P2X(7) receptor, and their ability to inhibit BzATP-induced IL-1beta release by differentiated THP-1 cells. Compounds 15a and 15d, with alkyl groups at the R(1) position, and especially compound 19h, with the 2-NO(2)-4,5-dimethoxy-benzyl group at the R(2) position, had potent inhibitory efficacy as P2X(7) antagonists.

The efflux pump inhibitor reserpine selects multidrug-resistant Streptococcus pneumoniae strains that overexpress the ABC transporters PatA and PatB.

One way to combat multidrug-resistant microorganisms is the use of efflux pump inhibitors (EPIs). Spontaneous mutants resistant to the EPI reserpine selected from Streptococcus pneumoniae NCTC 7465 and R6 at a frequency suggestive of a single mutational event were also multidrug resistant. No mutations in pmrA (which encodes the efflux protein PmrA) were detected, and the expression of pmrA was unaltered in all mutants. In the reserpine-resistant multidrug-resistant mutants, the overexpression of both patA and patB, which encode ABC transporters, was associated with accumulation of low concentrations of antibiotics and dyes. The addition of sodium orthovanadate, an inhibitor of ABC efflux pumps, or the insertional inactivation of either gene restored wild-type antibiotic susceptibility and wild-type levels of accumulation. Only when patA was insertionally inactivated were both multidrug resistance and reserpine resistance lost. Strains in which patA was insertionally inactivated grew significantly more slowly than the wild type. These data indicate that the overexpression of both patA and patB confers multidrug resistance in S. pneumoniae but that only patA is involved in reserpine resistance. The selection of reserpine-resistant multidrug-resistant pneumococci has implications for analogous systems in other bacteria or in cancer.

Parallel topology of genetically fused EmrE homodimers.

EmrE is a small H+-coupled multidrug transporter in Escherichia coli. Claims have been made for an antiparallel topology of this homodimeric protein. However, our own biochemical studies performed with detergent-solubilized purified protein support a parallel topology of the protomers. We developed an alternative approach to constrain the relative topology of the protomers within the dimer so that their activity can be assayed also in vivo before biochemical handling. Tandem EmrE was built with two identical monomers genetically fused tail to head (C-terminus of the first to N-terminus of the second monomer) with hydrophilic linkers of varying length. All the constructs conferred resistance to ethidium by actively removing it from the cytoplasm. The purified proteins bound substrate and transported methyl viologen into proteoliposomes by a proton-dependent mechanism. A tandem where one of the essential glutamates was replaced with glutamine transported only monovalent substrates and displayed a modified stoichiometry. The results support a parallel topology of the protomers in the functional dimer. The implications regarding insertion and evolution of membrane proteins are discussed.

In vitro infection model characterizing the effect of efflux pump inhibition on prevention of resistance to levofloxacin and ciprofloxacin in Streptococcus pneumoniae.

The prevalence of fluoroquinolone-resistant Streptococcus pneumoniae is slowly rising as a consequence of the increased use of fluoroquinolone antibiotics to treat community-acquired pneumonia. We tested the hypothesis that increased efflux pump (EP) expression by S. pneumoniae may facilitate the emergence of fluoroquinolone resistance. By using an in vitro pharmacodynamic infection system, a wild-type S. pneumoniae strain (Spn-058) and an isogenic strain with EP overexpression (Spn-RC2) were treated for 10 days with ciprofloxacin or levofloxacin in the presence or absence of the EP inhibitor reserpine to evaluate the effect of EP inhibition on the emergence of resistance. Cultures of Spn-058 and Spn-RC2 were exposed to concentration-time profiles simulating those in humans treated with a regimen of ciprofloxacin at 750 mg orally once every 12 h and with regimens of levofloxacin at 500 and 750 mg orally once daily (QD; with or without continuous infusions of 20 microg of reserpine/ml). The MICs of ciprofloxacin and levofloxacin for Spn-058 were both 1 microg/ml when susceptibility testing was conducted with each antibiotic alone and with each antibiotic in the presence of reserpine. For Spn-RC2, the MIC of levofloxacin alone and with reserpine was also 1 mug/ml; the MICs of ciprofloxacin were 2 and 1 microg/ml, respectively, when determined with ciprofloxacin alone and in combination with reserpine. Reserpine, alone, had no effect on the growth of Spn-058 and Spn-RC2. For Spn-058, simulated regimens of ciprofloxacin at 750 mg every 12 h or levofloxacin at 500 mg QD were associated with the emergence of fluoroquinolone resistance. However, the use of ciprofloxacin at 750 mg every 12 h and levofloxacin at 500 mg QD in combination with reserpine rapidly killed Spn-058 and prevented the emergence of resistance. For Spn-RC2, levofloxacin at 500 mg QD was associated with the emergence of resistance, but again, the resistance was prevented when this levofloxacin regimen was combined with reserpine. Ciprofloxacin at 750 mg every 12 h also rapidly selected for ciprofloxacin-resistant mutants of Spn-RC2. However, the addition of reserpine to ciprofloxacin therapy only delayed the emergence of resistance. Levofloxacin at 750 mg QD, with and without reserpine, effectively eradicated Spn-058 and Spn-RC2 without selecting for fluoroquinolone resistance. Ethidium bromide uptake and efflux studies demonstrated that, at the baseline, Spn-RC2 had greater EP expression than Spn-058. These studies also showed that ciprofloxacin was a better inducer of EP expression than levofloxacin in both Spn-058 and Spn-RC2. However, in these isolates, the increase in EP expression by short-term exposure to ciprofloxacin and levofloxacin was transient. Mutants of Spn-058 and Spn-RC2 that emerged under suboptimal antibiotic regimens had a stable increase in EP expression. Levofloxacin at 500 mg QD in combination with reserpine, an EP inhibitor, or at 750 mg QD alone killed wild-type S. pneumoniae and strains that overexpressed reserpine-inhibitable EPs and was highly effective in preventing the emergence of fluoroquinolone resistance in S. pneumoniae during therapy. Ciprofloxacin at 750 mg every 12 h, as monotherapy, was ineffective for the treatment of Spn-058 and Spn-RC2. Ciprofloxacin in combination with reserpine prevented the emergence of resistance in Spn-058 but not in Spn-RC2, the EP-overexpressing strain.

A quantitative method for routine measurement of cell surface P2X7 receptor function in leucocyte subsets by two-colour time-resolved flow cytometry.

The P2X(7) receptor is a ligand-gated cation channel activated by extracellular ATP and highly expressed on monocytes, macrophages and lymphocytes. Activation of this receptor by exposure to extracellular ATP opens a selective cation channel that allows Ca(2+) and Ba(2+) influx, and K(+) efflux. Over the first minute the channel adopts a second and larger permeability state allowing the uptake of ethidium(+), followed by a cascade of intracellular downstream effects. Current methods used to study the P2X(7) receptor function, do not give quantitative measurement in sub-populations of a mixed cell suspension. We describe a quantitative method to determine the P2X(7) receptor function using time-resolved two-colour flow cytometry by assessing ATP-induced ethidium(+) uptake. Practical factors such as ethidium bromide concentration, agonists, temperature and buffers are also studied. Moreover, the ATP-induced ethidium(+) uptake method is compared to ATP induced barium (Ba(2+)) influx with Fura-Red. These two compatible methods can be used to screen the channel/pore function of the cell surface P2X(7) receptor among individuals and the results may be useful to estimate susceptibility of subjects to certain infectious diseases.

Superoxide (*O2- ) production in CA1 neurons of rat hippocampal slices exposed to graded levels of oxygen.

Neuronal signaling, plasticity, and pathologies in CA1 hippocampal neurons are all intimately related to the redox environment and, thus tissue oxygenation. This study tests the hypothesis that hyperoxic superfusate (95% O(2)) causes a time-dependent increase in superoxide anion (*O(2)(-)) production in CA1 neurons in slices, which will decrease as oxygen concentration is decreased. Hippocampal slices (400 microm) from weaned rats were incubated with the fluorescent probe dihydroethidium (DHE), which detects intracellular *O(2)(-) production. Slices were loaded for 30 min using 10 microM DHE and maintained using one-sided superfusion or continuously loaded using 2.5 microM DHE and maintained using two-sided superfusion (36 degrees C). Continuous loading of DHE and two-sided superfusion gave the highest temporal resolution measurements of *O(2)(-) production, which was estimated by the increase in fluorescence intensity units (FIUs) per minute (FIU/min +/- SE) over 4 h. Superoxide production (2.5 microM DHE, 2-sided superfusion) was greatest in 95% O(2) (6.6 +/- 0.4 FIU/min) and decreased significantly during co-exposure with antioxidants (100 microM melatonin, 25 microM MnTMPyP) and lower levels of O(2) (60, 40, and 20% O(2) at 5.3 +/- 0.3, 3.3 +/- 0.1, and 1.6 +/- 0.2 FIU/min, respectively). CA1 cell death after 4 h (ethidium homodimer-1 staining) was greatest in 95% O(2) and lowest in 40 and 20% O(2). CA1 neurons generated evoked action potentials in 20% O(2) for >4 h, indicating viability at lower levels of oxygenation. We conclude that .O(2)(-) production and cell death in CA1 neurons increases in response to increasing oxygen concentration product (= PO(2) x time). Additionally, lower levels of oxygen (20-40%) and antioxidants should be considered to minimize superoxide-induced oxidative stress in brain slices.