Biomedical subjects
Publications and source records attributed to N E Allen.
LY173013 and LY186826 are bicyclic pyrazolidinones containing a novel aza-gamma-lactam ring structure. The antibacterial properties of these compounds appear to be related to those of beta-lactam antibiotics in that both classes of compounds share certain common binding molecules such as beta-lactamases and penicillin-binding proteins.
Vancomycin resistance in Enterococcus faecium 180, a clinical isolate from England, was studied. Resistance to vancomycin was transferable by conjugation to other enterococci. Expression of resistance was inducible and coincided with the appearance of a new membrane protein.
Forty-five aminoglycoside antibiotics and related compounds were compared for their ability to induce the accumulation of dihydrostreptomycin in Escherichia coli K12. The common aminoglycosides and a streptothricin antibiotic all induced enhanced uptake within a relatively narrow concentration range. These concentrations were lethal to the bacteria. Comparison of aminoacyl derivatives of tobramycin and apramycin, the latter synthesized utilizing transition-metal cations to selectively control the site of substitution, revealed that 1-N-aminoacyl modifications resulted in an increased ability to induce enhanced uptake. 2'-N-Aminoacyl modifications were also effective at inducing enhanced uptake, albeit without noticeable improvement over parent. The findings from this structure-activity comparison support the proposition that aminoglycosides share a common critical target (most likely the ribosome), which, when acted upon, results in both drug accumulation and killing.
LY146032, a cyclic lipopeptide antibiotic, is an inhibitor of cell wall peptidoglycan biosynthesis in gram-positive bacteria. Although LY146032 at relatively high concentrations inhibited the in vitro polymerization of UDP-linked sugar precursors, inhibition of cell wall formation in intact Staphylococcus aureus and Bacillus megaterium cells did not lead to the accumulation of UDP-N-acetyl-muramyl (MurNAc)-peptide(s). Experiments that measured formation of UDP-MurNAc-peptides revealed that LY146032 inhibited the formation of these nucleotide-linked intermediates. This antibiotic had a disruptive effect on membrane permeability as evidenced by the loss of intracellular potassium immediately after exposure to the drug. The lack of any major disruption of the phosphoenolpyruvate:sugar phosphotransferase system indicated that the membrane is not likely a lethal target for this antibiotic. The findings are consistent with a mechanism by which LY146032 inhibits the formation of precursor molecules utilized in peptidoglycan biosynthesis. The observed membrane effects likely result from transit of the inhibitor to its lethal target site.
Novel bacterial resistance genes were cloned and expressed as dominant selection markers in mammalian cells. Escherichia coli genes coding for resistance to the aminocyclitol antibiotics hygromycin B (Hm) and G418 were cloned into the eukaryotic expression plasmid pSV5GPT [Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78 (1981) 2072-2076]. Mouse cells normally sensitive to 100 micrograms/ml Hm were transformed with these plasmids and selected in 200 micrograms/ml Hm. Transformants resistant to as much as 1 mg/ml Hm and 500 micrograms/ml G418 were isolated. Cell extracts contained an acetyltransferase activity capable of acetylating G418 and an Hm aminocyclitol phosphotransferase activity. Plasmid DNA sequences were identified by Southern blot analysis of high Mr DNA isolated from transformed cells.
A plasmid conferring resistance to the aminocyclitol antibiotic hygromycin B was isolated from Escherichia coli. The gene conferring resistance to this drug was cloned in pBR322, and the gene was localized to a fragment of ca. 1,510 base pairs. Resistance to hygromycin B is determined by an aminocyclitol phosphotransferase that modifies hygromycin B and structurally related antibiotics. The specific modification of hygromycin B is a phosphorylation of the hydroxyl on the 4 position of the cyclitol ring (hyosamine). The presence of the phosphotransferase in E. coli correlates with reduced accumulation of [14C]hygromycin B.
Aminoglycoside-2"-O-adenylyltransferase was inhibited by 7-hydroxytropolone. Inhibition was competitive with respect to the cosubstrate ATP and appeared to require the unique vicinal arrangement of oxygens found in 7-hydroxytropolone. Combinations of 7-hydroxytropolone plus the appropriate aminoglycoside substrates were active against resistant bacteria possessing the adenylyltransferase. No potentiation was observed against other aminoglycoside-resistant or -susceptible strains. The fact that the inhibition of an aminoglycoside-modifying enzyme overcomes the poor uptake of aminoglycosides in resistant strains points to the singular importance of the inactivating enzyme as a determinant of resistance.
A novel low-molecular weight inhibitor of an aminoglycoside-inactivating enzyme, initially isolated from fermentation broths of Streptomyces neyagawaensis, was determined to be 7-hydroxytropolone. Its structure was confirmed by synthesis. In vitro synergy was demonstrated between 7-hydroxytropolone and certain aminoglycosides against bacteria which were resistant to those aminoglycosides by virtue of a 2"-O-adenylyltransferase. The synthesis and characterization of some analogs of 7-hydroxytropolone is also described.
Clinically isolated strains of Staphylococcus aureus that are inducibly resistant to both erythromycin and penicillin were susceptible to a combination of the two antibiotics. The synergistic effect of the combination results from an inhibition of penicillinase induction by erythromycin, sparing penicillin and allowing this drug to inhibit growth. When resistance to erythromycin is constitutive rather than inducible, the combination is no longer synergistic.
Induction of resistance to macrolide-, lincosamide-, and streptogramin B-type antibiotics in Staphylococcus aureus was studied by monitoring the appearance of erythromycin A (EM)-resistant [(14)C]leucine incorporation. Examination of the induction process revealed saturation kinetics and a time course much like that reported for penicillinase in gram-positive bacteria. Induction kinetics in exponentially growing cells were sigmoidal and appeared to reach a maximum and constant rate when growth reached stationary phase. Since the induction of EM-resistant colony-forming ability was complete within 60 min, ribosome modification cannot be limited to a fraction of the population and must occur in essentially every cell. However, EM-resistant growth was expressed in cells where less than half the [(14)C]leucine-incorporating activity was resistant to EM. This suggests that resistance requires that only a threshold level of ribosome modification be exceeded and that, once exceeded, resistance is dominant to sensitivity.
Several macrolide-, lincosamide-, and streptogramin B-type (MLS) antibiotics were tested as inducers of erythromycin A (EM)-resistant [(14)C]leucine incorporation. Only 14-membered-ring macrolides having a glycosidically linked 6-deoxy sugar at the C-3 position of the lactone ring and the structurally dissimilar lincosamide, celesticetin, showed inducer activity. Modifications of EM at the C-4'' position of cladinose can apparently destroy the inducer property but do not affect the inhibitory properties of the antibiotic. The findings clearly show that inducer and inhibitor activities can be dissociated and are consistent with the concept that distinct binding/receptor sites are utilized for inhibition of ribosome function and induction of resistance.
Explore the source record for details and available documents.
Target analyses of the induction of rho(-) mutants indicated that the cytoplasm of an aerobic nonrepressed yeast contains approximately 20 mutable units, whereas repressed cells exhibit approximately 3. Aerobic adaptation of fully repressed cells brings about an increase in these cytoplasmic units which continues after maximal respiration has been reached. This increase can be correlated with the increase in numbers of stained mitochondria. The data are interpreted with reference to mitochondrial deoxyribonucleic acid.