Aminoglycoside Uptake Research Articles

In vitro studies simultaneously examining effect of oxacillin on uptake of radiolabeled streptomycin and on associated bacterial lethality in Staphylococcus aureus.

We studied the effects of various concentrations of oxacillin on streptomycin uptake and killing for several strains of Staphylococcus aureus. When streptomycin was present in concentrations below the MIC, addition of oxacillin at concentrations greater than or equal to the MIC was associated with both significantly increased aminoglycoside uptake and killing. In contrast, when streptomycin was present in concentrations above the MIC, no increase of streptomycin uptake was noted with the addition of oxacillin, and killing was no greater than what would have been expected with a simply additive effect. Similar studies in a strain of S. aureus selected for high-level streptomycin resistance also demonstrated increased streptomycin uptake in the presence of concentrations of oxacillin above the MIC; however, killing was no greater than that seen with oxacillin alone. These studies provide data which are potentially important in designing a rational approach to clinical use of combination antibiotic therapy.

Involvement of outer membrane of Pseudomonas cepacia in aminoglycoside and polymyxin resistance.

Pseudomonas cepacia was found to be resistant to the outer membrane-permeabilizing effects of aminoglycoside antibiotics, polymyxin B, and EDTA. Permeabilization of P. cepacia to the fluorescent probe 1-N-phenylnaphthylamine was not achieved at concentrations 100- to 1,000-fold above those required to permeabilize Pseudomonas aeruginosa. Furthermore, in contrast to P. aeruginosa cells, intact cells of P. cepacia did not bind the fluorescent probe dansyl-polymyxin. However, purified lipopolysaccharide (LPS) from P. cepacia bound dansyl-polymyxin with approximately the same affinity as did LPS from P. aeruginosa. Also, binding of dansyl-polymyxin to P. cepacia (and P. aeruginosa) LPS was inhibited by polymyxin B, streptomycin, gentamicin, and Mg2+. These data suggest that P. cepacia does not utilize the self-promoted pathway for aminoglycoside uptake and that the outer membrane is arranged in a way that conceals or protects cation-binding sites on LPS which are capable of binding polycations such as aminoglycosides or polymxyin.

Penicillin-induced effects on streptomycin uptake and early bactericidal activity differ in viridans group and enterococcal streptococci.

In vitro studies with penicillin and [3H]streptomycin in four strains of streptococci (S. faecalis, S. sanguis, and S. mitis) were performed by simultaneously measuring the rates of bacterial killing and uptake of streptomycin. In S. faecalis, penicillin stimulated streptomycin uptake, as has been shown by Moellering and Weinberg (R. C. Moellering, Jr., and A. N. Weinberg, J. Clin. Invest. 50:2580-2584, 1971). Moreover, the antibiotic combination was associated with an enhanced bactericidal rate which temporally correlated with beta-lactam-induced aminoglycoside uptake. In contrast, in viridans group streptococci (S. sanguis and S. mitis) penicillin had no effect on streptomycin uptake and a minimal effect on bactericidal rate when compared with either drug alone. These data suggested that the stimulation of streptomycin uptake in streptococci by penicillin is strain or species specific and that important differences exist between enterococci and viridans group streptococci regarding the mechanisms of beta-lactam-aminoglycoside potentiation.

High-level amikacin resistance in Escherichia coli due to phosphorylation and impaired aminoglycoside uptake

Plasmid pMP1-1 in Escherichia coli L-0 encodes aminoglycoside (AG) 3'-phosphotransferase II [APH(3')-II]. This enzyme modifies and confers high-level resistance to kanamycin. Although amikacin is a substrate for APH(3')-II, strain L-0(pMP1-1) is susceptible to amikacin. Plasmid pMP1-2 is a spontaneous mutant of pMP1-1 which determines increased APH(3')-II activity for amikacin, apparently as a result of an increase in the copy number of the plasmid. From amikacin-susceptible, gentamicin-susceptible transformants and transconjugants that bear the APH(3')-II gene on plasmid pMP1-1 or pMP1-2 or cloned into multicopy plasmid pBR322, we selected spontaneous mutants at concentrations of amikacin or gentamicin that were two to four times higher than the MICs of these antibiotics. In each case, whether they were selected by using amikacin or gentamicin, the mutants exhibited modest (two- to eightfold) increases in the MIC of gentamicin and major (64- to 128-fold) increases in the MIC of amikacin. Using these laboratory strains of E. coli, we examined the effects on AG susceptibility of the interaction of AG-modifying enzyme activity and generalized AG uptake. Increasing the level of activity of an AG phosphotransferase in these strains lowered their susceptibility to AGs which were substrates for which the enzyme had low Kms. However, an increase in AG-modifying activity alone did not result in large increases in the MICs for poor substrates of the enzyme. In strains which lacked AG-modifying enzymes, a decrease in the rate of AG uptake increased the MICs modestly for a broad spectrum of AGs. When a strain bore the phosphotransferase, a decrease in generalized AG uptake could raise the MIC further, not only for low-Km substrates, but even for AG substrates for which the enzyme had high Kms. Thus, increased modifying activity, together with a diminished rate of uptake, could produce even higher MICs for poor AG substrates.

In-vitro uptake of gentamicin and tobramycin by rat renal tubules in the presence or absence of Escherichia coli endotoxin.

Renal tubules of rats were incubated with aminoglycoside (gentamicin or 3H-tobramycin, 10 mg/l) in the presence or absence of Escherichia coli endotoxin (10 mg/l). The kinetics of aminoglycoside uptake by the tubule were only slightly affected by endotoxin. The percentage of serum in the medium affected the tobramycin uptake. This uptake decreased from a mean ratio of concentration in the tubules/concentration in the medium (T/M) of 1.63 in 5% serum to a mean T/M of 0.86 in 10% serum (P less than 0.01).

The kinetics of dihydrostreptomycin uptake in Pseudomonas putida membrane vesicles: absence of inhibition by cations

Dihydrostreptomycin was taken up in isolated cytoplasmic membrane vesicles of Pseudomonas putida by an active transport mechanism. Saturation kinetics were observed with an apparent Km and Vmax of 15 mM and 50 nmol/min/mg of protein respectively. The evidence suggested that the observed kinetics was that of the energy-dependent phase I component of dihydrostreptomycin uptake. Neither magnesium nor the polyamine, spermine, inhibited dihydrostreptomycin transport. Thus, the inhibition of aminoglycoside uptake in intact cells of Gram-negative bacteria and the increase in the minimal inhibitory concentration in the presence of multivalent cations and polyamines were interpreted to be effects that take place at the outer membrane level.

Lack of accumulation of exogenous adenylyl dihydrostreptomycin by whole cells or spheroplasts of Escherichia coli

Accumulation of purified adenylylated dihydrostreptomycin (DHS-AMP) was examined in two strains of Escherichia coli. E. coli JSRO-N was plasmid free and aminoglycoside (AG) susceptible; E. coli JSRO-N(pSAD1) contained a plasmid-encoded AG adenylyltransferase which modifies DHS and streptomycin and confers resistance to both of these drugs. Although both whole cells and spheroplasts of JSRO-N accumulated free DHS, we were not able to demonstrate uptake of DHS-AMP by this strain. Whole cells and spheroplasts of JSRO-N(pSAD1) accumulated DHS at a much slower rate than that observed in JSRO-N. This was presumably due to the activity of the adenylyltransferase in JSRO-N(pSAD1). However, this low rate of accumulation of DHS was still higher than the uptake of DHS-AMP by either JSRO-N or JSRO-N(pSAD1). Thus, the rate of accumulation of DHS-AMP was even lower than that of DHS during the slow, initial, energy-dependent phase of AG uptake seen in JSRO-N(pSAD1). We also found that when either JSRO-N or JSRO-N(pSAD1) was incubated with barely inhibitory or subinhibitory concentrations of DHS, rapid uptake of DHS could be stimulated by the addition of an inhibitory concentration of another AG, such as amikacin. Uptake of DHS-AMP could not be similarly enhanced by the addition of amikacin. Our results indicate that DHS-AMP is not accumulated by whole cells or spheroplasts of E. coli. These results are consistent with the postulated intracellular location of AG-modifying enzymes.

Roles of ribosomal binding, membrane potential, and electron transport in bacterial uptake of streptomycin and gentamicin.

The effects of a set of conditions on aminoglycoside uptake were determined. Membrane vesicles either with a membrane potential (delta psi) of -125 mV (adequate to drive lysine uptake) or with succinate, lactate, or phenazine methosulfate did not accumulate gentamicin unless components of protein synthesis were included. Ribosomally resistant (rpsL) Escherichia coli cells demonstrated energy-dependent phase II uptake similar to that of a streptomycin-susceptible strain of E. coli when treated with 100 micrograms of puromycin per ml. Puromycin (100 micrograms/ml) also increased the uptake of the cationic compounds polyamine and arginine. These studies support a role of protein synthesis in aminoglycoside uptake and in the development of energy-dependent phase II. delta psi of cells did not increase either at the initiation of or during energy-dependent phase II, showing that energy-dependent phase II is not due to an elevation of delta psi. In a Bacillus subtilis system, significant streptomycin uptake requires a threshold value of delta psi which varies depending upon the concentration of streptomycin used. At 25 micrograms/ml, the uptake of streptomycin reached maximal levels after exceeding the threshold value, whereas at 100 micrograms/ml there was a gradual increase of the uptake to the maximal after the threshold value was exceeded. Several studies supported the view that electron transport has a specific role other than its requirement to produce the cellular delta psi. The uptake of gentamicin was stimulated to a greater extent by phenazine methosulfate-ascorbate than by the ionophore nigericin in strains of E. coli, although nigericin stimulated delta psi to a greater degree. Cells with 25% of the normal quinone concentration had delta psi values identical to cells with the normal quinone concentration, but the quinone-deficient cells had a significantly lower rate of gentamicin uptake. KCN prevented gentamicin uptake but did not prevent the development of delta psi. The effects of ubiquinone depletion in an E. coli strain were more evident on gentamicin uptake than on ATP-driven glutamine transport or proton motive force-driven proline transport, consistent with a specific requirement for quinones in aminoglycoside uptake. A detailed explanation of the mechanism of accumulation of streptomycin and gentamicin and a proposed mechanism for killing bacterial cells by these agents have been provided.

7-Hydroxytropolone: an inhibitor of aminoglycoside-2"-O-adenylyltransferase

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.

Role of the membrane potential in bacterial resistance to aminoglycoside antibiotics.

The electrical potential difference (delta psi) across the membrane of Escherichia coli was measured by the distribution of lipid-soluble cations and correlated with resistance to dihydrostreptomycin, where resistance is presumed due to reduced uptake of the drug. A good correlation between the two measured parameters was found under all conditions tested, which included effects of several mutations, inhibitors, changes in pH, and osmolarity. The most dramatic changes were seen when pH was varied; in wild-type strains resistance increased more than 100-fold, and delta psi fell by 70 mV when pH was reduced from 8.5 to 5.5. These results were interpreted as support for a model in which the uptake of the polycationic aminoglycosides is electrogenic and therefore driven by delta psi. The factor common to mutations and conditions which increase resistance was a reduction in delta psi. A simple model was developed which relates the minimal inhibitory concentration to the rate of aminoglycoside uptake and the rate of growth.

Involvement of the outer membrane in gentamicin and streptomycin uptake and killing in Pseudomonas aeruginosa.

Induction of a major outer membrane protein, H1, in Pseudomonas aeruginosa resulted in decreased susceptibility to gentamicin and streptomycin. Mutants which overproduce protein H1 and cells in which H1 is induced in response to growth conditions had altered kinetics of uptake and killing. It was further demonstrated that gentamicin and streptomycin interact with the outer membrane to permeabilize it to lysozyme and to increase the permeation of a chromogenic beta-lactam, nitrocefin. Experiments with inhibitors of aminoglycoside uptake showed that uptake was not required to increase permeability. Mg2+ at 1 mM totally inhibited aminoglycoside-mediated outer membrane permeabilization. We propose that the uptake and killing by these aminoglycosides requires interaction with an Mg2+ binding site at the outer membrane, permitting aminoglycoside uptake into the periplasm.

Aminoglycoside uptake and mode of action—with special reference to streptomycin and gentamicin

Aminoglycoside uptake and mode of action—with special reference to streptomycin and gentamicin

Aminoglycoside uptake and mode of action-with special reference to streptomycin and gentamicin. II. Effects of aminoglycosides on cells.

Aminoglycoside uptake and mode of action-with special reference to streptomycin and gentamicin. II. Effects of aminoglycosides on cells.

Interaction between aminoglycoside uptake and ribosomal resistance mutations.

Mutants resistant to the 2-deoxystreptamine aminoglycosides hygromycin B and gentamicin were analyzed biochemically and genetically. In hygromycin B-resistant strains, ribosomal alterations were not detectable by electrophoretic or genetic experiments. Rather, as was demonstrated for one strain in detail, resistance to this drug seems to be the consequence of several mutations, each impairing drug accumulation, namely of a deletion of a gene close to the proC marker which potentiates the effect of a second mutation in the unc gene cluster. Three mutants resistant to gentamicin which were previously demonstrated to harbor an altered ribosomal protein, L6, were shown in addition to contain unc. Both the unc and the ribosomal mutation greatly impair the drug accumulation ability of the mutants. Further evidence for the direct effect of ribosomal mutations on the uptake of aminoglycosides was obtained with strains that possess ribosomes with increased affinity for dihydrostreptomycin. Dihydrostreptomycin transport by these cells is greatly stimulated; thus, the hypersensitivity of these mutants is caused by increased binding affinity for dihydrostreptomycin and its secondary effect on the uptake process. Experiments were also performed on the biochemical basis of the third phase of aminoglycoside transport (acceleration phase). The condition for its onset is that ribosomes are active in protein synthesis irrespective of whether the proteins synthesized are functional. This, and the failure to observe the synthesis of new proteins upon the addition of aminoglycosides, do not support the view of autoinduction of a cognate or related transport system.

Gentamicin uptake in wild-type and aminoglycoside-resistant small-colony mutants of Staphylococcus aureus.

Gentamicin uptake and killing were studied in aminoglycoside-susceptible wild-type Staphylococcus aureus strains and aminoglycoside-resistant small-colony mutants selected by gentamicin from these strains. In wild-type S. aureus three phases of gentamicin accumulation were noted, and killing occurred during the last and most rapid phase of uptake. Uptake and killing were abolished by anaerobic growth and sodium azide, suggesting that energy-dependent active drug transport required respiration. Treatment of wild-type strains with the uncouplers N,N'-dicyclohexyl carbodiimide (DCCD) and carbonyl cyanide-m-chlorophenyl hydrazone showed disparate effects on gentamicin uptake, producing enhanced and diminished accumulations, respectively. Small-colony mutants demonstrated markedly deficient uptake compared with the wild-type strains and were not killed by gentamicin in concentrations up to 10 mug/ml. Several classes of aminoglycoside-resistant mutant strains are described. One mutant strain was a menadione auxotroph which, when grown in the presence of menadione, exhibited normal gentamicin uptake and killing. Gentamicin uptake and killing in this strain were abolished by KCN when the strain was grown in a medium supplemented with menadione. The membrane adenosine triphosphatase inhibitor DCCD was lethal for this mutant but not for other mutants or wild-type strains. Preincubation with menadione prevented the lethal effect of DCCD, and this strain demonstrated normal gentamicin accumulation when exposed to both DCCD and menadione. A second mutant strain demonstrated both gentamicin uptake and killing in the presence but not the absence of DCCD. Studies with small-colony mutants of S. aureus indicated that the defect in aminoglycoside uptake is very likely related to an inability to generate or maintain energized membranes from respiration. These studies suggest that the membrane energization associated with active aminoglycoside accumulation requires electron transport for the generation of a protonmotive force.

Clinical isolation and characterization of aminoglycoside-resistant small colony variants of enterobacter aerogenes

Small colony variants of Enterobacter aerogenes, as well as the parental large colony type, grew in blood drawn for cultures on three separate days from a patient who had received suboptimal gentamicin therapy. Minimum inhibitory concentrations of four aminoglycoside antibiotics were eight to more than 16 times higher for small colony variants than for the normal large colony type. Small colony variants had defective catalase activity, which may have interfered with oxidative metabolism and aminoglycoside uptake. Small colony variants reverted readily to the parental type in vitro in the absence of aminoglycosides. Clinically isolated small colony variants appeared similar to those selected in the presence of gentamicin in vitro, with respect to colony morphology, aminoglycoside resistance and catalase deficiency. The isolation of small colony variants during gentamicin therapy in vivo suggests that such variants may be a cause of treatment failure in patients receiving aminoglycosides.