Search PubMed⌕ Search

Biomedical subjects

S B Levy

Publications and source records attributed to S B Levy.

At least 55 records · Page 3Linked to original sources
PubMed1998

Association of organic solvent tolerance and fluoroquinolone resistance in clinical isolates of Escherichia coli.

Fluoroquinolone resistance in Escherichia coli is principally caused by two kinds of mutation: those affecting the target proteins of the drugs, i.e. DNA gyrase and topoisomerase IV, and those affecting regulatory genes such as marA, soxS or robA. Recently, overexpression of the latter genes was linked to increased organic solvent tolerance in E. coli. Among 138 clinical fluoroquinolone-resistant and -susceptible clinical isolates of E. coli we found a high association between fluoroquinolone resistance and organic solvent tolerance. This finding suggests that E. coli may undergo an adaptive response to extrinsic substances other than quinolones, while mutating to fluoroquinolone resistance.

Anti-Infective Agents↗
PubMed1998

Overexpression of the marA or soxS regulatory gene in clinical topoisomerase mutants of Escherichia coli.

The contribution of regulatory genes to fluoroquinolone resistance was studied with clinical Escherichia coli strains bearing mutations in gyrA and parC and with different levels of fluoroquinolone resistance. Expression of marA and soxS was evaluated by Northern blot analysis of isolates that demonstrated increased organic solvent tolerance, a phenotype that has been linked to overexpression of marA, soxS, and rob. Among 25 cyclohexane-tolerant strains detected by a screen for increased organic solvent tolerance (M. Oethinger, W. V. Kern, J. D. Goldman, and S. B. Levy, J. Antimicrob. Chemother. 41:111-114, 1998), we found 5 Mar mutants and 4 Sox mutants. A further Mar mutant was detected among 11 fluoroquinolone-resistant, cyclohexane-susceptible E. coli strains used as controls. Comparison of the marOR sequences of clinical Mar mutants with that of E. coli K-12 (GenBank accession no. M96235) revealed point mutations in marR in all mutants which correlated with loss of repressor function as detected in a marO::lacZ transcriptional assay. We found four other amino acid changes in MarR that did not lead to loss of function. Two of these changes, present in 20 of the 35 sequenced marOR fragments, identified a variant genotype of marOR. Isolates with the same gyrA and parC mutations showed increased fluoroquinolone resistance when the mutations were accompanied by overexpression of marA or soxS. These data support the hypothesis that high-level fluoroquinolone resistance involves mutations at several chromosomal loci, comprising structural and regulatory genes.

Anti-Infective Agents↗
PubMed1998

Multidrug resistance following expression of the Escherichia coli marA gene in Mycobacterium smegmatis.

Expression of the Escherichia coli multiple antibiotic resistance marA gene cloned in Mycobacterium smegmatis produced increased resistance to multiple antimicrobial agents, including rifampin, isoniazid, ethambutol, tetracycline, and chloramphenicol. Cloned marR or marA cloned in the antisense direction had no effect. Resistance changes were lost with spontaneous loss of the plasmid bearing marA. A MarA mutant protein, having an insertional mutation within either of its two alpha-helices of the first putative helix-turn-helix domain, failed to produce the multiresistance phenotype in E. coli and M. smegmatis, indicating that this region is critical for MarA function. These results strongly suggest that E. coli marA functions in M. smegmatis and that a mar-like regulatory system exists in this organism.

Bacterial Proteins↗
PubMed1997

Emergence of different mechanisms of resistance in the evolution of multidrug resistance in murine erythroleukemia cell lines.

We examined the genetic and biochemical bases for drug resistance and the order of appearance of different mechanisms underlying the increasingly more resistant murine erythroleukemia cell lines established in Adriamycin (ADR). In the first-step low-level resistant cell line PC4-A5 (able to grow in 5 ng/mL ADR), there was a 2-fold reduction in topoisomerase IIalpha and topoisomerase IIbeta mRNA levels, as well as topoisomerase IIalpha protein and activity levels as compared with the parental cell line. The topoisomerase IIalpha activity levels remained reduced as the cells became increasingly more resistant. In contrast, the topoisomerase II mRNA and protein levels returned to approximately the parental levels in resistant cells growing in higher drug concentrations (40-160 ng/mL). Parental cells expressed the multidrug resistance protein (MRP), but beginning with PC4-A5 MRP expression decreased and remained reduced in increasingly resistant cell lines. At high levels of ADR resistance, the cells expressed the mdr3 gene concomitant with the appearance of vincristine resistance and energy-dependent daunomycin and vincristine efflux. Glutathione levels, internal pH, and expression of the major vault protein (MVP) remained unchanged in all cell lines. Fluorescence microscopy revealed no alterations in daunomycin distribution or vesicle numbers between the parental and resistant cell lines. Different resistance mechanisms emerge sequentially as cells become more resistant to ADR; the mechanisms are retained during the development of multidrug resistance (MDR). In intermediate-level MDR cell lines (PC4-A10 and PC4-A20), resistance involves an as yet undetermined mechanism(s).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Active efflux of the free acid form of the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in multidrug-resistance-protein-overexpressing murine and human leukemia cells.

Murine and human cell lines overexpressing the multidrug-resistance protein (MRP) showed a marked decreased accumulation of the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In contrast, less altered accumulation was seen in the P-glycoprotein(P-gp)-overexpressing cell lines. The decreased drug accumulation was reversed by the energy inhibitors sodium azide/2-deoxyglucose and by the vinca alkaloid, vincristine, but not by the chemotherapeutic agents, etoposide and adriamycin. Decreased accumulation was linked to active efflux of the hydrophilic free acid form of BCECF from the MRP-overexpressing cell lines, indicating that dye extrusion occurs after the dye ester has been converted to the free acid form in the cytoplasm. The finding suggests that MRP mediates removal of substrates from a cytoplasmic location. Buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, decreased the vincristine and etoposide resistance displayed by the MRP-expressing murine cell lines, but did not affect the accumulation of BCECF. Thus, while glutathione may be involved in MRP-mediated resistance to some chemotherapeutic agents, it is not necessary for effiux of substrates such as BCECF.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Antibiotic resistance: an ecological imbalance.

Antibiotic resistance thwarts the treatment of infectious diseases worldwide. Although a number of factors can be identified which contribute to the problem, clearly the antibiotic as a selective agent and the resistance gene as the vehicle of resistance are the two most important, making up a 'drug resistance equation'. Both are needed in order for a clinical problem to arise. Given sufficient time and quantity of antibiotic, drug resistance will eventually appear. But a public health problem is not inevitable if the two components of the drug resistance equation are kept in check. Enhancing the emergence of resistance is the case by which resistance determinants and resistant bacteria can spread locally and globally, selected by widespread use of the same antibiotics in people, animal husbandry and agriculture. Antibiotics are societal drugs. Each individual use contributes to the sum total of society's antibiotic exposure. In a broader sense, the resistance problem is ecological. In the framework of natural competition between susceptible and resistant bacteria, antibiotic use has encouraged growth of the resistant strains, leading to an imbalance in prior relationships between susceptible and resistant bacteria. To restore efficacy to earlier antibiotics and to maintain the success of new antibiotics that are introduced, we need to use antibiotics in a way which assures an ecological balance that favours the predominance of susceptible bacterial flora.

Animals↗
PubMed1997

Indomethacin-mediated reversal of multidrug resistance and drug efflux in human and murine cell lines overexpressing MRP, but not P-glycoprotein.

Decreased accumulation of the fluorescent dye BCECF [2', 7'-bis-(2-carboxyethyl)-5-(6)- carboxyfluorescein] characterized murine and human multidrug-resistant cell lines overexpressing the multidrug resistance protein (MRP). Indomethacin (10 microM), a known cyclo-oxygenase and glutathione-S-transferase inhibitor as well as a modulator of anion transport, increased accumulation and blocked efflux of BCECF in MRP-expressing murine and human cells. The drug did not affect P-glycoprotein (P-gp)-mediated export of rhodamine 123. The indomethacin effect on BCECF efflux was not reversed by the addition of exogenous prostaglandins, suggesting that the drug acts by a mechanism other than decreasing prostaglandin synthesis. Indomethacin also increased multidrug susceptibility of both murine and human cell lines overexpressing MRP, but not those displaying P-gp-associated resistance. In addition, indomethacin modulated the decreased vincristine accumulation in cells expressing MRP, but not in those expressing P-gp. These data suggest that indomethacin is a specific inhibitor of MRP, possibly functioning by inhibition of glutathione-S-transferase or, alternatively, by direct competition with the drug at the transport site.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Effect of glucose transport inhibitors on vincristine efflux in multidrug-resistant murine erythroleukaemia cells overexpressing the multidrug resistance-associated protein (MRP) and two glucose transport proteins, GLUT1 and GLUT3.

The relationship between mammalian facilitative glucose transport proteins (GLUT) and multidrug resistance was examined in two vincristine (VCR)-selected murine erythroleukaemia (MEL) PC4 cell lines. GLUT proteins, GLUT1 and GLUT3, were constitutively coexpressed in the parental cell line and also in the VCR-selected cell lines. Increased expression of the GLUT1 isoform was noted both in the PC-V40 (a non-P-glycoprotein, mrp-overexpressing subline) and in the more resistant PC-V160 (overexpressing mrp and mdr3) cell lines. Overexpression of GLUT3 was detected only in the PC-V160 subline. An increased rate of facilitative glucose transport (Vmax) and level of plasma membrane GLUT protein expression paralleled increased VCR resistance, active VCR efflux and decreased VCR steady-state accumulation in these cell lines. Glucose transport inhibitors (GTIs), cytochalasin B (CB) and phloretin blocked the active efflux and decreased steady-state accumulation of VCR in the PC-V40 subline. GTIs did not significantly affect VCR accumulation in the parental or PC-V160 cells. A comparison of protein sequences among GLUT1, GLUT3 and MRP revealed a putative cytochalasin B binding site in MRP, which displayed 44% sequence similarity/12% identity with that previously identified in GLUT1 and GLUT3; these regions also exhibited a similar hydropathy plot pattern. The findings suggested that CB bound to MRP and directly or indirectly lowered VCR efflux and/or CB bound to one or both GLUT proteins, which acted to lower the VCR efflux mediated by MRP. This is the first report of a non-neuronal murine cell line that expressed GLUT3.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Inhibition of the multiple antibiotic resistance (mar) operon in Escherichia coli by antisense DNA analogs.

The multiple antibiotic resistance operon (marORAB) in Escherichia coli controls intrinsic susceptibility and resistance to multiple, structurally different antibiotics and other noxious agents. A plasmid construct with marA cloned in the antisense direction reduced LacZ expression from a constitutively expressed marA::lacZ translational fusion and inhibited the induced expression of LacZ in cells bearing the wild-type repressed fusion. The marA antisense construction also decreased the multiple antibiotic resistance of a Mar mutant. Two antisense phosphorothioate oligonucleotides, one targeted to marO and the other targeted to marA of the mar operon, introduced by heat shock or electroporation reduced LacZ expression in the strain having the marA::lacZ fusion. One antisense oligonucleotide, tested against a Mar mutant of E. coli ML308-225, increased the bactericidal activity of norfloxacin. These studies demonstrate the efficacy of exogenously delivered antisense oligonucleotides targeted to the marRAB operon in inhibiting expression of this chromosomal regulatory locus.

Cloning, Molecular↗
PubMed1997

Selection of multiple-antibiotic-resistant (mar) mutants of Escherichia coli by using the disinfectant pine oil: roles of the mar and acrAB loci.

Mutants of Escherichia coli selected for resistance to the disinfectant pine oil or to a household product containing pine oil also showed resistance to multiple antibiotics (tetracycline, ampicillin, chloramphenicol, and nalidixic acid) and overexpressed the marA gene. Likewise, antibiotic-selected Mar mutants, which also overexpress marA, were resistant to pine oil. Deletion of the mar or acrAB locus, the latter encoding a multidrug efflux pump positively regulated in part by MarA, increased the susceptibility of wild-type and mutant strains to pine oil.

Ampicillin↗
PubMed1997

Role of the acrAB locus in organic solvent tolerance mediated by expression of marA, soxS, or robA in Escherichia coli.

Escherichia coli K-12 strains are normally tolerant to n-hexane and susceptible to cyclohexane. Constitutive expression of marA of the multiple antibiotic resistance (mar) locus or of the soxS or robA gene product produced tolerance to cyclohexane. Inactivation of the mar locus or the robA locus, but not the soxRS locus, increased organic solvent susceptibility in the wild type and Mar mutants (to both n-hexane and cyclohexane). The organic solvent hypersusceptibility is a newly described phenotype for a robA-inactivated strain. Multicopy expression of mar, soxS, or robA induced cyclohexane tolerance in strains with a deleted or inactivated chromosomal mar, soxRS, or robA locus; thus, each transcriptional activator acts independently of the others. However, in a strain with 39 kb of chromosomal DNA, including the mar locus, deleted, only the multicopy complete mar locus, consisting of its two operons, produced cyclohexane tolerance. Deletion of acrAB from either wild-type E. coli K-12 or a Mar mutant resulted in loss of tolerance to both n-hexane and cyclohexane. Organic solvent tolerance mediated by mar, soxS, or robA was not restored in strains with acrAB deleted. These findings strongly suggest that active efflux specified by the acrAB locus is linked to intrinsic organic solvent tolerance and to tolerance mediated by the marA, soxS, or robA gene product in E. coli.

Bacterial Proteins↗
PubMed1997

Glutamate residues located within putative transmembrane helices are essential for TetA(P)-mediated tetracycline efflux.

The tetA(P) gene from Clostridium perfringens encodes a unique membrane protein that is responsible for the active efflux of tetracycline from resistant cells. The novel TetA(P) protein has neither the typical structure nor the conserved motifs that are found in tetracycline efflux proteins from classes A through H or classes K and L. Site-directed mutagenesis of selected residues within TetA(P) was performed to elucidate their role in tetracycline efflux. Glutamate residues 52 and 59, negatively charged residues located within putative transmembrane helix 2, could not be replaced by either glutamine or aspartate and so were essential for tetracycline efflux. Replacement of Glu89, which was located at the end of helix 3, by aspartate but not by glutamine allowed TetA(P) function, indicating the importance of a carboxyl group at this position. After mutation of the Asp67 residue, located within cytoplasmic loop 1, no immunoreactive protein was detected. It is concluded that negatively charged residues that appear to be located within or near the membrane are important for the function of TetA(P).

Antiporters↗