In vitro susceptibility of Pythium insidiosum isolates to aminoglycoside antibiotics and tigecycline

This study evaluated the in vitro activity of aminoglycoside antibiotics and tigecycline against Pythium insidiosum. The susceptibility tests were carried out using the broth microdilution method in accordance with the CLSI document M38-A2. MIC values for gentamicin, neomycin, paromomycin, and streptomycin ranged from 32 to 64 mg/liter, and the minimal fungicidal concentration (MFC) ranged from 32 to 128 mg/liter, which are incompatible with safe concentrations of these drugs in plasma in vivo. Tigecycline showed the lowest MIC (0.25 to 2 mg/liter) and MFC (1 to 8 mg/liter) range values. The in vitro susceptibility observed to tigecycline makes this drug a good option in future tests in vitro and in vivo for the management of pythiosis.     

The influence of aminoglycoside antibiotics on the in vitro function of rat liver ribosomes

There are few studies of the influence of aminoglycoside antibiotics on the ribosomes of higher eukaryotic organisms. To this end, cytoplasmic ribosomes were prepared from rat liver. In vitro, poly(U)-directed ribosome protein synthesis was studied in the presence and absence of selected aminoglycosides. Misreading of poly(U) was also assessed. Consistent with earlier studies using different sources of ribosomes, paromomycin inhibited cell-free protein synthesis and caused poly(U) misreading. In contrast to the findings of other studies in cell-free ribosomes of eukaryotic organisms, netilmicin, tobramycin, and neomycin were most active in inhibiting protein synthesis, and gentamicin C2 and neomycin caused appreciable misreading. Thus the previous suggestion that a paromamine fragment (found in paromomycin) might be a structural requirement for in vitro inhibition of protein synthesis and misreading is not substantiated by the results in rat liver ribosomes. Commercial gentamicin C is a mixture of gentamicins C1, C1a, and C2. Despite nearly identical chemical structures, the three constituents displayed greatly different propensities for inducing poly(U) misreading. C2 was the most active, followed by C1a. In summary, selected aminoglycoside antibiotics caused inhibition and mistranslation of poly(U) messenger in an in vitro ribosome system prepared from rat liver. These effects were not limited to paromamine-containing aminoglycoside antibiotics. Gentamicin C2 caused much more poly(U) misreading than the other two constituents of the gentamicin C complex.     

Practical computer-assisted dosing for aminoglycoside antibiotics.

In principle, computer-assisted individualization of antibiotic dosing offers the prospect of better patient outcomes through improved dosing precision. In practice, however, the expertise in pharmacokinetics required to operate these programs has precluded their use by most physicians and pharmacists. We developed a computer program for individualization of dosing of aminoglycoside antibiotics under conditions in which access to experts in pharmacokinetics is impractical. The program is accurate, yet it requires less effort for data collection than previous drug dosing programs did. The program generates advice on a broad spectrum of topics, including dose adjustment, interpretation of measured drug concentrations in blood, and recommendations for monitoring drug concentrations. We tested its performance by prospectively comparing it with a clinical pharmacokinetic consultation service in a series of 78 consecutive patients. There were no differences in accuracy or bias in the prediction of drug concentrations. The rate of agreement between the program's dosing recommendations and those of the consultation service was 67 percent. This rate of agreement is typical of interexpert variation. In a stratified set of 24 of the 41 instances with significant disagreement regarding the recommended dose, experts ranked the program's recommendations as highly as those of the consultation service (95% confidence interval for difference in rank, -0.30 less than chi less than 0.47). The results suggest that expert systems can be coupled with pharmacokinetic dosing programs to deliver high-quality clinical recommendations for administration of antimicrobial agents.     

Comparative pharmacokinetics of aminoglycoside antibiotics in guinea pigs.

The pharmacokinetics of netilmicin, gentamicin, and tobramycin in plasma and in perilymph of guinea pigs were studied after a single intravenous injection of 40 mg/kg. Detailed pharmacokinetic analysis of the plasma drug concentration-time data up to 36 h after the intravenous dose revealed that the pharmacokinetics of the aminoglycoside antibiotics can be best described as a three-compartment open model. The disposition half-lives (t1/2) in plasma of the three antibiotics were comparable and within the following ranges: t1/2 alpha of 0.09 to 0.16 h; t1/2 beta of 0.88 to 1.01 h; and t1/2 gamma of 7.87 to 8.29 h. The volume of distribution in the central compartment and the total body clearance of netilmicin (294 ml/kg, 5.74 ml/min per kg) were greater than those of gentamicin (160 ml/kg, 3.40 ml/min per kg) and tobramycin (204 ml/kg, 4.63 ml/min per kg). Pharmacokinetic analysis of the perilymph drug concentration-time data indicated that all three antibiotics penetrated the perilymph readily, but netilmicin cleared from the perilymph compartment faster than gentamicin and tobramycin. The maximum perilymph drug concentrations were 4.17, 8.05, and 6.78 micrograms/ml and occurred at 1, 2, and 4 h for netilmicin, gentamicin, and tobramycin, respectively. The ratio of area under the curve of perilymph to plasma was lowest for netilmicin (0.27), followed by gentamicin (0.39) and tobramycin (0.57). These results suggest that the differences in pharmacokinetics and concentrations of netilmicin in the perilymph may account for less ototoxic liability of netilmicin compared with gentamicin and tobramycin.     

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.     

Inhibition of DNA replication initiation by aminoglycoside antibiotics.

The reinitiation of DNA replication induced by a temperature shift in a dnaC(Ts) mutant of Escherichia coli was markedly inhibited by aminoglycoside antibiotics around the MIC in a short period. Protein synthesis continued for several minutes after the addition of aminoglycosides but was immediately blocked by chloramphenicol, suggesting that the inhibition of initiation of replication by aminoglycosides is not a secondary effect due to the interruption of protein synthesis. Aminoglycosides did not significantly affect RNA synthesis, suggesting that primer RNA synthesis for DNA initiation is not blocked by the agents. The lethal action of habekacin was observed simultaneously with the inhibition of DNA reinitiation. DNA elongation demonstrated with a dnaE(Ts) mutant or toluene-treated cells of a polA mutant was not significantly affected by aminoglycosides. The oriC-membrane complex formation was markedly interrupted by habekacin in the dnaC(Ts) mutant, and the in vitro reconstitution of the oriC-membrane complex was completely blocked by aminoglycosides. The present studies show that aminoglycosides block initiation of DNA replication and suggest that the inhibition is caused by the interruption of oriC-membrane attachment.     

In vitro and in vivo antibacterial activities of dactimicin, a novel pseudodisaccharide aminoglycoside, compared with those of other aminoglycoside antibiotics

When compared with astromicin, amikacin, gentamicin, and sisomicin, dactimicin was similar to astromicin in in vitro activity and was more active than amikacin and gentamicin against the clinical isolates of Serratia marcescens, but less active against Pseudomonas aeruginosa. Dactimicin and astromicin were active against many gentamicin- and amikacin-resistant bacteria expressing aminoglycoside-modifying enzymes, with the exception of aminoglycoside 3-acetyltransferase. However, dactimicin was more resistant than astromicin to inactivation by aminoglycoside 3-acetyltransferase, probably owing to the protective action of the formimidoyl group. The in vivo activity of dactimicin, assessed by the 50% effective doses against systemic infections in mice, was similar or superior to that of astromicin and was superior or inferior to that of amikacin depending on the strains tested.     

Long-term protection of polyaspartic acid in experimental gentamicin nephrotoxicity.

Polyaspartic Acid (PAA) protects the kidney from experimental gentamicin nephrotoxicity despite large increases in renal cortical gentamicin content. In these experiments, prominent cytoplasmic vacuoles were noted in all animals that received PAA with or without gentamicin. The present study showed that there were no renal structural or functional consequences of PAA given alone or with gentamicin for up to 14 days, followed by a 16-week washout period. Creatinine clearance was similar to that of controls in animals that received gentamicin and in those that received PAA alone. Thus, complete functional protection was conferred by PAA and gentamicin, confirming previous reports from our laboratory. There was no protection by PAA from the nephrotoxic effects of mercuric chloride and cis-platinum.     

Mechanisms of action of aminoglycoside antibiotics in eucaryotic protein synthesis.

Tetrahymena thermophila is a eucaryotic organism that is highly susceptible to growth inhibition by aminoglycoside antibiotics. Concentrations of paromomycin, gentamicin G418, and hygromycin B at 22, 10, and 17 microM, respectively, inhibited growth by 50%. A combination of in vitro and in vivo methods was used to determine the mechanisms of action of these aminoglycoside antibiotics on protein synthesis in T. thermophila. Analysis of polysome profiles from paromomycin- and gentamicin G418-treated cells showed clear, progressive depletions of polysomes concomitant with an inhibition of in vivo [14C] lysine incorporation. In vitro, paromomycin and gentamicin G418, which are disubstituted 2-deoxystreptamine-containing molecules, were not very effective inhibitors of either the translocation of peptidyl-tRNA or the elongation of nascent polypeptide chains on polysomes. In contrast, we found that the translocation of phe-tRNA on polyuridylate programmed ribosomes was susceptible to inhibition by paromomycin. We conclude that the primary inhibitory action of paromomycin and gentamicin G418 was at (i) an early stage of elongation after initiation, (ii) the initiation stage of translation, or (iii) a stage of translation before initiation. Hygromycin B, which is a monosubstituted 2-deoxystreptamine-containing aminoglycoside, potently inhibited the elongation of nascent chains during the translation of polysomes. In addition, the in vitro translation of polysomes from two hygromycin B-resistant mutants was resistant to the inhibition of elongation caused by hygromycin B.     

Modifications of aminoglycoside antibiotics targeting RNA

The increased awareness of the central role of RNA has led to realization that RNA, as structural and functional information accumulation, is also drug target to small molecular therapy. Aminoglycosides are a group of well-known antibiotics, which function through binding to specific sites in prokaryotic ribosomal RNA (rRNA) and affecting the fidelity of protein synthesis. Unfortunately, their clinical practice has been curtailed by toxicity and rapid increasing number of resistant strains. Therefore, it is highly desirable to design new modified aminoglycosides that will overcome the undesirable properties of natural occurring aminoglycosides. On the other hand, aminoglycosides as potential antiviral (HIV) agents were also reported. Herein, we survey the current efforts to develop new aminoglycoside derivatives with modification and reconstruction on each sugar ring and review the latest advances in structure-activity relationships (SAR).     

Determination of aminoglycoside antibiotics in biological fluids: a review.

We describe current procedures for measuring aminoglycoside antibiotics. Although many of these techniques are suitable for clinical adaptation, selection of a method for use in a clinical laboratory will depend on the budget and availability of equipment and expertise.     

Effects of aminoglycoside antibiotics on polymorphonuclear leukocyte function in vivo.

In vitro incubation of aminoglycoside antibiotics with human polymorphonuclear leukocytes (PMNs) has been shown to induce abnormalities in cell function. This study was designed to determine whether there are similar abnormalities in leukocyte function after exposure to the action of these agents in vivo. Four aminoglycosides (gentamicin, tobramycin, netilmicin, and amikacin) were tested. In vitro incubation did not induce a chemotactic defect when measured by an under-agarose method. However, inhibition of candidacidal activity was reproducible after in vitro incubation of all aminoglycosides tested. Nevertheless, when the aminoglycosides were administered intravenously to normal volunteers, PMN function, including adherence to nylon wool columns, chemotaxis, phagocytosis, and killing of Candida albicans, was unimpaired at 1, 3, and 24 h postinfusion. Therefore, we conclude that aminoglycoside antibiotic administration does not induce PMN dysfunction in vivo.     

Synergism at clinically attainable concentrations of aminoglycoside and beta-lactam antibiotics.

We evaluated the in vitro synergistic activity at clinically attainable concentrations of combinations of aminoglycoside and beta-lactam antibiotics against 30 gentamicin-resistant clinical isolates of gram-negative bacilli. All 56 pairs of 4 aminoglycosides and 14 beta-lactams were evaluated. Combinations with amikacin demonstrated inhibitory synergistic activity in 29% of the assays, as compared with 22% for netilmicin (P = 0.018), 17% for gentamicin (P less than 0.001), and 13% for tobramycin (P less than 0.001). Among the beta-lactams, combinations with cefoperazone, ceftriaxone, or cefpiramide (SM-1652) demonstrated inhibitory synergistic activity most often (39, 38, and 35% of the assays, respectively) and with ceforanide, cefsulodin, and imipenem least often (less than or equal to 8% each). The most active combination was amikacin and ceftriaxone, with which 67% of the assays demonstrated inhibitory synergism. Isolates with high-level resistance to either antibiotic in a combination were unlikely to be inhibited synergistically by the combination. Further, combinations generally demonstrated little synergistic activity against isolates highly susceptible to beta-lactams.     

Stimulatory and inhibitory properties of aminoglycoside antibiotics at N-methyl-D-aspartate receptors.

The effects of aminoglycoside antibiotics on N-methyl-D-aspartate (NMDA) receptors were studied using voltage-clamp recording of recombinant NMDA receptors expressed in Xenopus oocytes. A number of aminoglycosides were found to potentiate macroscopic currents at heteromeric NR1A/NR2B receptors, but not at NR1A/NR2A, NR1A/NR2C, NR1A/NR2D, or NR1B/NR2B receptors. The degree of potentiation had a rank order neomycin B > paromomycin > gentamicin C > geneticin > kanamycin A > streptomycin. Potentiation was not seen with kasugamycin and spectinomycin. The degree of stimulation paralleled the number of the amino groups in the aminoglycosides. The stimulatory effects of aminoglycosides were more pronounced at subsaturating concentrations of glycine and at acidic pH, similar to the stimulatory effects of spermine. We measured the effects of aminoglycosides at mutant NMDA receptors to determine which amino acid residues in NMDA receptor subunits are involved in stimulation. Mutations that reduced or abolished spermine stimulation also reduced stimulation by aminoglycosides. Several aminoglycosides produced a weak voltage-dependent block of NMDA receptors, but the degree of inhibition did not appear to correlate with the number of amino groups in the molecule. The results suggest that aminoglycosides having more than three amino groups have stimulatory effects that are mediated through the spermine-binding site on NMDA receptors.     

Saturday Conference: a clinician looks at the aminoglycoside antibiotics.

Aminoglycoside antibiotics provide physicians with valuable drugs for coping with serious, often hospital-acquired gram-negative rod infections which are occurring with increasing frequency. Use of these agents may be advantageous and even lifesaving. There are potential hazards associated with their administration. Narrow therapeutic indices coupled with susceptibility to degradation by resistant bacteria should temper our enthusiasm for aminoglycosides.