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Biomedical subjects

S B Levy

Publications and source records attributed to S B Levy.

At least 37 records · Page 2Linked to original sources
PubMed2000

'Intergenic' blr gene in Escherichia coli encodes a 41-residue membrane protein affecting intrinsic susceptibility to certain inhibitors of peptidoglycan synthesis.

In the annotation of genomic sequences, small open reading frames (ORFs) are often neglected, particularly if they have no homology to other ORFs or proteins. A mini-TnphoA insertion in a 602 bp 'intergenic' region of the Escherichia coli chromosome at genomic nucleotide 1702674 gave rise to a membrane-bound PhoA fusion protein and a two- to fourfold increase in the intrinsic susceptibility to a wide spectrum of beta-lactam antibiotics without affecting beta-lactamase activity or susceptibility to tetracycline, chloramphenicol, gentamicin or quinolones. Susceptibility was also increased to cycloserine and bacitracin, but not to fosfomycin or valinomycin; these drugs, like beta-lactams, inhibit peptidoglycan synthesis, although by different mechanisms. A clone bearing only 358 bp of this 'blr' region restored resistance to the parental level. Two amber mutations in the clone prevented such restoration and were counteracted by an amber suppressor, proving that the active species is a protein. The Blr protein has 41 amino acids, with a single predicted transmembrane helix, but no clear homology to any other protein. A transcriptional start exists 39 bp upstream from the translational start. The membrane location of Blr suggests that it may be part of an efflux pump or involved in murein metabolism. The results indicate that genes for other very small functional proteins may lie within 'intergenic' regions.

Alkaline Phosphatase↗
PubMed2000

The quarternary molecular architecture of TetA, a secondary tetracycline transporter from Escherichia coli.

TetA, a tetracycline cation/proton antiporter, was expressed in Escherichia coli with a C-terminal tag of six histidines, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography. Two-dimensional crystals were obtained after reconstitution of purified protein with lipids. Electron microscopy of negatively stained crystals revealed a trigonal symmetry, from which we infer that this secondary transporter has a trimeric structure. An overall molecular envelope can be described by a triangle of side approximately 100 A enclosing a central stain-filled depression. These dimensions are consistent with those obtained from projection views of single, isolated TetA particles that also display a trimeric architecture, confirming that the threefold symmetry is not simply a consequence of crystal-packing interactions. These data represent the first direct view of the quarternary arrangement of any antibiotic efflux pump. They are fully consistent with biochemical data on TetA, which indicate that it functions as a multimer and that the monomer consists of two domains, one of which plays the major part in oligomerization interactions.

Antiporters↗
PubMed2000

Antibiotic and antiseptic resistance: impact on public health.

More and more we are moving patients from hospitals to homes for continued treatment. Vancomycin and triclosan were used for 30 years before any resistance emerged, because their applications were strictly limited. Today, after greatly increased use, resistance to both antibiotics and antibacterials has appeared. Of importance there are genetic links between resistance to antibiotics and to antibacterials. Health professionals and the public need to be educated about the rational use of drugs that affect the microbial world. The Alliance for the Prudent Use of Antibiotics, an international organization established in 1981 with members in more than 100 countries, has adopted education as its prime mission. Via its web site (www.apua.org) and linked information on reservoirs of antibiotic resistance (ROAR) among nonpathogenic bacteria, it reaches both providers and consumers. The message is simple: bacteria are needed for our survival. The vast majority of bacteria perform important functions that are crucial for our lives. Prudent use of both antibiotics and antibacterials must be championed to achieve and maintain the balanced microbial environment in which we have entered and evolved.

Bacteria↗
PubMed2000

Ineffectiveness of topoisomerase mutations in mediating clinically significant fluoroquinolone resistance in Escherichia coli in the absence of the AcrAB efflux pump.

Fluoroquinolone-resistant mutants, selected from a wild-type Escherichia coli K-12 strain and its Mar mutant by exposure to increasing levels of ofloxacin on solid medium, were analyzed by Northern (RNA) blot analysis, sequencing, and radiolabelled ciprofloxacin accumulation studies. Mutations in the target gene gyrA (DNA gyrase), the regulatory gene marR, and additional, as yet unidentified genes (genes that probably affect efflux mediated by the multidrug efflux pump AcrAB) all contributed to fluoroquinolone resistance. Inactivation of the acrAB locus made all strains, including those with target gene mutations, hypersusceptible to fluoroquinolones and certain other unrelated drugs. These studies indicate that, in the absence of the AcrAB pump, gyrase mutations fail to produce clinically relevant levels of fluoroquinolone resistance.

Anti-Infective Agents↗
PubMed2000

Non-target gene mutations in the development of fluoroquinolone resistance in Escherichia coli.

Mutations in loci other than genes for the target topoisomerases of fluoroquinolones, gyrA and parC, may play a role in the development of fluoroquinolone resistance in Escherichia coli. A series of mutants with increasing resistance to ofloxacin was obtained from an E. coli K-12 strain and five clinical isolates. First-step mutants acquired a gyrA mutation. Second-step mutants reproducibly acquired a phenotype of multiple antibiotic resistance (Mar) and organic solvent tolerance and showed enhanced fluoroquinolone efflux. None of the second-step mutants showed additional topoisomerase mutations. All second-step mutants showed constitutive expression of marA and/or overexpressed soxS. In some third-step mutants, fluoroquinolone efflux was further enhanced compared to that for second-step mutants, even when the mutant had acquired additional topoisomerase mutations. Attempts to circumvent the second-step Mar mutation by induction of the mar locus with sodium salicylate and thus to select for pure topoisomerase mutants at the second step were not successful. At least in vitro, non-target gene mutations accumulate in second- and third-step mutants upon exposure to a fluoroquinolone and typically include, but do not appear to be limited to, mutations in the mar or sox regulons with consequent increased drug efflux.

Anti-Infective Agents↗
PubMed2000

Differential expression of over 60 chromosomal genes in Escherichia coli by constitutive expression of MarA.

In Escherichia coli, the MarA protein controls expression of multiple chromosomal genes affecting resistance to antibiotics and other environmental hazards. For a more-complete characterization of the mar regulon, duplicate macroarrays containing 4,290 open reading frames of the E. coli genome were hybridized to radiolabeled cDNA populations derived from mar-deleted and mar-expressing E. coli. Strains constitutively expressing MarA showed altered expression of more than 60 chromosomal genes: 76% showed increased expression and 24% showed decreased expression. Although some of the genes were already known to be MarA regulated, the majority were newly determined and belonged to a variety of functional groups. Some of the genes identified have been associated with iron transport and metabolism; other genes were previously known to be part of the soxRS regulon. Northern blot analysis of selected genes confirmed the results obtained with the macroarrays. The findings reveal that the mar locus mediates a global stress response involving one of the largest networks of genes described.

Bacterial Proteins↗
PubMed2000

Second-site suppressor mutations of inactivating substitutions at gly247 of the tetracycline efflux protein, Tet(B).

An Asp or Asn substitution for Gly247 in transmembrane helix 8 (TM-8) of Tet(B), the tetracycline efflux protein, eliminated tetracycline resistance. Second site suppressor mutations which partially restored resistance were located in TM-5, -8, -10, or -11 or in cytoplasmic loop 8-9 or loop 10-11. These results indicate physical proximity or functional relationships between TM-8 and these other regions of Tet(B).

Amino Acid Substitution↗
PubMed1999

The mar regulon: multiple resistance to antibiotics and other toxic chemicals.

The chromosomal multiple antibiotic resistance (mar) locus of Escherichia coli and other members of the Enterobacteriaceae controls resistance to multiple, structurally unrelated compounds including antibiotics, household disinfectants, organic solvents and other toxic chemicals. The Mar phenotype is induced following exposure to a variety of chemicals with aromatic rings.

Anti-Bacterial Agents↗
PubMed1999

Genetic evidence that InhA of Mycobacterium smegmatis is a target for triclosan.

Three Mycobacterium smegmatis mutants selected for resistance to triclosan each had a different mutation in InhA, an enoyl reductase involved in fatty acid synthesis. Two expressed some isoniazid resistance. A mutation originally selected on isoniazid also mediated triclosan resistance, as did the wild-type inhA gene on a multicopy plasmid. Replacement of the mutant chromosomal inhA genes with wild-type inhA eliminated resistance. These results suggest that M. smegmatis InhA, like its Escherichia coli homolog FabI, is a target for triclosan.

Anti-Infective Agents, Local↗
PubMed1999

Nomenclature for new tetracycline resistance determinants.

Letters of the English alphabet have heretofore been used to name tetracycline resistance determinants. Since all 26 letters have now been used, a nomenclature employing numerals is recommended for future determinants, and one laboratory has offered to coordinate the assignment of numerals.

Terminology as Topic↗
PubMed1999

Reversal of tetracycline resistance mediated by different bacterial tetracycline resistance determinants by an inhibitor of the Tet(B) antiport protein.

Active efflux is a useful strategy by which bacteria evade growth inhibition by antibiotics. Certain semisynthetic tetracycline (TC) analogs, substituted at the 13th carbon at C-6 on ring C of the TC molecule, blocked TC efflux as revealed in everted membrane vesicles from class B TC-resistant (Tcr) Escherichia coli (M. L. Nelson, B. H. Park, J. S. Andrews, V. A. Georgian, R. C. Thomas, and S. B. Levy, J. Med. Chem. 36:370-377, 1993). A representative C-13-substituted analog, 13-cyclopentylthio-5-OH-TC (13-CPTC), was shown to competitively inhibit TC translocation by the Tet(B) protein, blocking the uptake of TC into vesicles and therefore the efflux of TC from whole cells. Against Tcr E. coli, 13-CPTC, when used in combination with doxycycline, produced synergistic inhibition of growth. 13-CPTC was shown to increase the uptake of [3H]TC into the resistant cells. 13-CPTC alone was a potent growth inhibitor against TC-susceptible (Tcs) and Tcr Staphylococcus aureus and enterococci specifying class K or class L efflux-dependent TC resistance mechanisms or, unexpectedly, the class M ribosomal protection mechanism. These findings indicate that derivatives of TC, identified by their ability to block the Tet(B) efflux protein, can restore TC activity against Tcr bacteria bearing either of the two known resistance mechanisms. Blocking drug efflux and increasing intracellular drug concentrations constitute an effective approach to reversing TC resistance and may be generally applicable to other antibiotics rendered ineffective by efflux proteins.

Bacteria↗
PubMed1999

Characterization of MarR superrepressor mutants.

MarR negatively regulates expression of the multiple antibiotic resistance (mar) locus in Escherichia coli. Superrepressor mutants, generated in order to study regions of MarR required for function, exhibited altered inducer recognition properties in whole cells and increased DNA binding to marO in vitro. Mutations occurred in three areas of the relatively small MarR protein (144 amino acids). It is surmised that superrepression results from increased DNA binding activities of these mutant proteins.

Bacterial Proteins↗
PubMed1999

Alteration of the repressor activity of MarR, the negative regulator of the Escherichia coli marRAB locus, by multiple chemicals in vitro.

MarR negatively regulates expression of the multiple antibiotic resistance operon (marRAB) in Escherichia coli. In this study, it was demonstrated that sodium salicylate, plumbagin, 2, 4-dinitrophenol, and menadione-inducers of the marRAB operon in whole cells-all interfered with the repressor activity of MarR in vitro. It is proposed that these compounds can interact directly with MarR to affect its repressor activity.

2,4-Dinitrophenol↗
PubMed1999

Release of bacteria during the purge cycles of steam-jacketed sterilizers.

The design of the steam-jacketed sterilizer includes an exterior air-gap fixture through which purged chamber aerosols potentially could escape into the ambient environment. Studies of the purge cycle in two sterilizer models tested the potential release of a genetically marked Enterococcus faecalis, together with Bacillus stearothermophilus spores introduced as exposed cultures. Direct plate counts, broth enrichment and polymerase chain reaction analysis were used to confirm any released organisms trapped in an all-glass impinger. From the retrieval of both bacterial strains, an estimated 10(3) organisms can be released from uncontained bacterial loads of 10(11) E. faecalis and 10(7) B. stearothermophilus, even from properly functioning autoclaves. The release of an opportunistic pathogen from sterilizer purge exhausts emphasises the importance of proper sterilizer location, ventilation, containment of heavily contaminated loads, and adequate sterilizer maintenance.

Air Microbiology↗
PubMed1998

Overexpression of marA, soxS, or acrAB produces resistance to triclosan in laboratory and clinical strains of Escherichia coli.

Triclosan (Irgasan) is a broad spectrum antimicrobial agent used in handsoaps, toothpastes, fabrics, and plastics. It inhibits lipid biosynthesis in Escherichia coli, probably by action upon enoyl reductase (FabI) (McMurry, L.M., Oethinger, M. and Levy, S.B. (1988) Nature 394, 531-532). We report here that overexpression of the multidrug efflux pump locus acrAB, or of marA or soxS, both encoding positive regulators of acrAB, decreased susceptibility to triclosan 2-fold. Deletion of the acrAB locus increased the susceptibility to triclosan approximately 10-fold. Four of five clinical E. coli strains which overexpressed marA or soxS also showed enhanced triclosan resistance. The acrAB locus was involved in the effects of triclosan upon both cell growth rate and cell lysis.

Anti-Infective Agents, Local↗