Structural and phylogenetic analyses of resistance to next-generation aminoglycosides conferred by AAC(2') enzymes.

Plazomicin is currently the only next-generation aminoglycoside approved for clinical use that has the potential of evading the effects of widespread enzymatic resistance factors. However, plazomicin is still susceptible to the action of the resistance enzyme AAC(2')-Ia from Providencia stuartii. As the clinical use of plazomicin begins to increase, the spread of resistance factors will undoubtedly accelerate, rendering this aminoglycoside increasingly obsolete. Understanding resistance to plazomicin is an important step to ensure this aminoglycoside remains a viable treatment option for the foreseeable future. Here, we present three crystal structures of AAC(2')-Ia from P. stuartii, two in complex with acetylated aminoglycosides tobramycin and netilmicin, and one in complex with a non-substrate aminoglycoside, amikacin. Together, with our previously reported AAC(2')-Ia-acetylated plazomicin complex, these structures outline AAC(2')-Ia's specificity for a wide range of aminoglycosides. Additionally, our survey of AAC(2')-I homologues highlights the conservation of residues predicted to be involved in aminoglycoside binding, and identifies the presence of plasmid-encoded enzymes in environmental strains that confer resistance to the latest next-generation aminoglycoside. These results forecast the likely spread of plazomicin resistance and highlight the urgency for advancements in next-generation aminoglycoside design.

[ThE COMPARATIVE CHARACTERISTIC OF PHENOTYPIC AND GENOTYPIC RESISTANCE TO AMINOGLYCOSIDES OF STRAINS STAPHYLOCOCCUS AUREUS ISOLATED IN TRAUMATOLOGICAI ORTHOPEDIC HOSPITAL].

The clinical isolates of Staphylococcus aureus (n = 102) were analyzed on sensitivity and to gentamicin, tobramicin, netimicin and amikacin. The disc diffusing technique was applied. The technique ofpolymerase chain reaction was applied to analyze all strains establishing presence in their genomes genes aac (6'-Ie/aph(2"), ant1, aac, ant(6)-Ia, aph (3')-IIIa and ant(4')-Ia coding amino-glycoside-modifying enzymes. The strains sensitive to amino-glycosides had no the given genes in genome. The genome of all strains resistant to amino-glycosides included no less than two of enumerated genes. The 100% correlation was established between phenotypic resistance of analyzed strains to amino-glycosides and availability in them of gene aac(6')-Ie/aph(2").

Cosubstrate tolerance of the aminoglycoside resistance enzyme Eis from Mycobacterium tuberculosis.

We previously demonstrated that aminoglycoside acetyltransferases (AACs) display expanded cosubstrate promiscuity. The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis is responsible for the resistance of this pathogen to kanamycin A in a large fraction of clinical isolates. Recently, we discovered that Eis is a unique AAC capable of acetylating multiple amine groups on a large pool of aminoglycoside (AG) antibiotics, an unprecedented property among AAC enzymes. Here, we report a detailed study of the acyl-coenzyme A (CoA) cosubstrate profile of Eis. We show that, in contrast to other AACs, Eis efficiently uses only 3 out of 15 tested acyl-CoA derivatives to modify a variety of AGs. We establish that for almost all acyl-CoAs, the number of sites acylated by Eis is smaller than the number of sites acetylated. We demonstrate that the order of n-propionylation of the AG neamine by Eis is the same as the order of its acetylation. We also show that the 6' position is the first to be n-propionylated on amikacin and netilmicin. By sequential acylation reactions, we show that AGs can be acetylated after the maximum possible n-propionylation of their scaffolds by Eis. The information reported herein will advance our understanding of the multiacetylation mechanism of inactivation of AGs by Eis, which is responsible for M. tuberculosis resistance to some AGs.

Impact of metal ions on netilmicin-melanin interaction.

Netilmicin, which is mainly used as the sulfate, is a semisynthetic, water soluble aminoglycoside antibiotic obtained by chemical modification of sisomicin. It is active against both Gram-positive and Gram-negative bacteria, including strains which are resistant to other aminoglycosides. Netilmicin form complexes with melanin. The aim of the presented work was to examine the effect of Cu2+, Zn2+, Ca2+ and Mg2+ on netilmicin binding to synthetic DOPA-melanin. It has been demonstrated that metal ions decrease the amount of antibiotic bound to melanin as compared with netilmicin-melanin complexes obtained in the absence of metals. It has been also shown that only one class of binding sites participates in netilmicin-[melanin-metal ion] complexes formation with the association constant K approximately 10(3) M(-1). The obtained results demonstrate that Cu2+, Zn2+, Ca2+ and Mg2+ ions modify the interaction between netilmicin and melanin biopolymer. The blocking of some active centers in melanin molecules by metal ions, which potentially exist in living systems, may influence the clinical therapeutic efficiency as well as the undesirable side effects of netilmicin.

[The role of aminoglycoside--modifying enzymes in resistance of Gram-negative rods]. / Prawdopodobny udzial enzymów paleczek Gram-ujemnych w profilu opornosci na antybiotyki aminoglikozydowe.

The aim of the study was to evaluate the aminoglycoside resistance of Gram-negative bacilli isolated from patients. To the examination 35 strains of Enterobacteriaceae and 18 of non-fermentative bacteria were included. Resistance to aminoglycosides (gentamicin (G), netilmicin (Nt), tobramycin (T), amikacin (A), kanamycin (K), neomycin (N)) was established by disk diffusion method. Interpretation of enzymatic mechanisms was performed by Livermore. The most common enzymes AAC(6')I were found in Enterobacteriaceae group (mostly in E. cloaceae and P. mirabilis) and AAC(3') and in non-fermentative bacteria: AAC(6')I in P. aeruginosa and APH(3')VI and AAC(3')I in A. baumanii. The most frequent phenotype was resistance to six antibiotics (G, Nt, T, A, K, N) Resistance rates were high for gentamicin (>70 %) in both groups and amikacin (88,89 %) in non-fermentatives.

Influence of flow rate on the disposition of levofloxacin and netilmicin in the isolated rat lung.

The objective of this study was to examine the influence of the rate at which the tissue is perfused on the disposition of levofloxacin and netilmicin in the pulmonary tissue, using an experimental model of the isolated rat lung. Analysis of the results was performed using two pharmacokinetic approaches. By stochastic analysis of outflow curves the corresponding statistical moments and derived distribution coefficient were calculated. Model-dependent analysis based on a three-compartment dispersion model was also applied to the outflow concentration data. A statistically significant decrease in the distribution coefficient was observed for both antibiotics when the flow rate was decreased. For levofloxacin this parameter takes values of 2.14 and 1.25 mL/g for 10 and 5 mL/min flow rates, respectively. In the case of netilmicin these values were 0.81 and 0.39 mL/g for the higher and lower flow rates, respectively. Model parameters related to the distribution process were also modified as a consequence of the flow rate decrease. Tissue flow rate seems to be a determinant factor on the distribution of levofloxacin and netilmicin in the isolated rat lung.

Study of aminoglycoside-nucleic acid interactions by an HPLC method.

The interactions of a number of aminoglycoside antibiotics with tRNA and DNA were studied by an HPLC method. based on tRNA and DNA peak size exclusion. Among the compounds studied (deoxystreptamine, neamine, neomycin B, kanamycin A, gentamicin A, netilmicin, streptomycin, and the synthetic neamine analogue BKN3), neomycin B and the synthetic analogue of neamine were proved to be the most potent binders.

The inductive role of ionic binding in the bactericidal and postexposure effects of aminoglycoside antibiotics with implications for dosing.

Gram-negative bacilli precooled to 4 degrees C to inactivate energy-dependent drug transport were exposed to an aminoglycoside antibiotic to assess the antibacterial effect of passive ionic binding of drug. Removal of free drug and energizing the cells by incubation at 37 degrees C showed the postexposure effect to be bactericidal. The effect was directly related to the amount and concentration of drug in the initial exposure medium proportional to the bacterial density. Binding was nonsaturable at the highest drug:bacteria ratio tested. Elution, exposure of spheroplasts, and inhibition by divalent cations indicated binding sites in the outer bacterial membrane. Different bacterial species had variable efficiency but similar patterns of binding different aminoglycosides reflecting in vitro susceptibility. The self-promoted postexposure internalization of ionically bound aminoglycoside accounts for the early drug-concentration-dependent rapid bactericidal action of aminoglycosides. The phenomenon has implications for effective initial dosing with aminoglycoside antibiotics.

Adaptive resistance to aminoglycoside antibiotics from first-exposure down-regulation.

Adaptive resistance to the bactericidal effect of an aminoglycoside antibiotic was induced in Pseudomonas aeruginosa and other aerobic gram-negative bacilli by initial exposure to the drug. Both subinhibitory and inhibitory concentrations produced resistance in bacterial cells surviving the effects of the initial ionic binding. Development of drug refractoriness required an adaptive period of growth, was enhanced by the continued presence of drug, and reversed after several hours of growth in drug-free medium. Unstable resistance was not explained by selection of mutants. The mechanism of adaptive resistance was down-regulation of aminoglycoside uptake during the period of accelerated energy dependent drug transport (EDP II). Down-regulation induced by gentamicin or tobramycin produced cross-resistance to other aminoglycosides. The kinetics of unstable first-exposure resistance suggests that a continuous drug level might not provide the most effective therapy with aminoglycosides and gives rationale to larger initial and longer interval bolus dosing.

Acute prostatitis: which antibiotic to use first.

Six antimicrobial agents were administered to 48 patients (divided in 6 groups) who underwent prostatectomy. Half of the patients received the antibiotic in a single dose one hour before the operation and the rest in divided doses 24 hours before the operation. The concentration levels in serum and in prostatic tissue were measured for each of the antibiotics and for each mode of administration. The obtained ratios of prostatic tissue to serum concentrations and the relative antimicrobial activity to local pathogens of each agent indicate that the agent of choice for prostatic disease is netilmicin followed by aztreonam, cefuroxime and the ticarcillin-clavulanic acid combination.

Comparative study with enoxacin and netilmicin in a pharmacodynamic model to determine importance of ratio of antibiotic peak concentration to MIC for bactericidal activity and emergence of resistance.

An in vitro pharmacokinetic model was used to study the comparative antibacterial activities of multiple-dose regimens of enoxacin and netilmicin. Strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus were exposed to changing drug concentrations, mimicking human two-compartment pharmacokinetics. Oral administration was simulated for the quinolone, and intravenous administration was simulated for the aminoglycoside. Similar ratios of peak concentration to MIC resulted in similar changes in bacterial concentrations over time with both compounds. Following the initial dose, a rapid bactericidal effect occurred, with a greater than 99% reduction of the bacterial counts within 4 h at peak concentrations more than three times the MIC. However, bacterial regrowth occurred within 24 h unless the peak concentration/MIC ratio exceeded 8:1 (P less than 0.01). For the regrowing bacteria, MICs were four- to eightfold higher, and little or no bactericidal effect occurred following the second and subsequent doses. These data demonstrate the equally potent bactericidal activity of orally administered enoxacin and intravenously administered netilmicin. Selection of resistant subpopulations was similar with each drug. The peak concentration/MIC ratio may be an important parameter in the clinical use of quinolone and aminoglycoside antibiotics.

Antibiotic penetration into the infected knee. A rabbit experiment.

We investigated the diffusion of penicillin-G, cloxacillin, clindamycin, and netilmicin into synovial fluid and membrane in rabbits. Purulent arthritis was induced in the right knee of each rabbit by inoculation of Staphylococcus aureus phage type 3C, whereas sterile saline was injected into the left knee to serve as a control. Two days later, concentrations of antibiotics were determined in serum, synovial fluid, and membrane after an intramuscular single dose. All four drugs diffused readily into infected joints, whereas the corresponding concentrations in the normal joints were 2-3 times lower. Clindamycin showed the highest intraarticular penetration, cloxacillin the lowest. The lower penetration of cloxacillin corresponded to its higher protein binding in rabbit serum. Considering the sufficient local concentrations achieved, parenteral treatment obviates the need for local instillation of these antibiotics.

Experimental pneumococcus infection in mice: correlation of bactericidal activity in vitro with the effect in vivo for gentamicin, netilmicin and tobramycin.

An experimental model in mice, incorporating the intraperitoneal inoculation of a Streptococcus pneumoniae type 3, was used to evaluate the effect in vivo after single-dose administration of the three aminoglycosides, gentamicin, tobramycin and netilmicin, and to correlate this effect with their in vitro activity against the pathogen, in particular the bactericidal rate. The minimal inhibitory concentrations (MIC's), which were equal to the minimal bactericidal concentrations (MBC's), were 12.5 micrograms/ml for netilmicin, and 25 micrograms/ml for the two other aminoglycosides, respectively. All three antibiotics showed excellent bactericidal activities even at concentrations 1/4 times the MIC's, but the bactericidal rate was clearly lower for tobramycin than for the two other aminoglycosides. The effect in vivo measured as the 50% effective dose (ED50) closely reflected the relative bactericidal activities of the drugs. Of the pharmacokinetic parameters investigated on dosages equal to the ED50's for the three drugs, the best to correlate with the bactericidal rates in vitro were the peak serum concentrations.

Renal handling of netilmicin in the rat with streptozotocin-induced diabetes mellitus.

We examined the hypothesis that the reduced accumulation of aminoglycoside in renal cortex of rats with streptozotocin-induced diabetes mellitus (DM) is secondary to lower rates of tubular transport of drug compared with non-DM rats. Using whole kidney clearance techniques we found that the fractional excretion of [3H]netilmicin in DM rats rose from an initial value of 92.4 +/- 1.3% to 101 +/- 4.5% (N = 10) after eight 20-min periods. These values were not significantly different from those of non-DM rats (96.4 +/- 1.8 and 106.7 +/- 1.4%, respectively, N = 7). The plasma concentration of drug was similar in the two groups whereas inulin clearance and the filtered load of drug were higher in DM rats. In both groups microinjection experiments revealed the presence of an absorptive flux of [3H]netilmicin along the proximal tubule and loop of Henle, but no absorptive flux was detected along the distal nephron. In free-flow micropuncture experiments in DM rats a net secretory flux of netilmicin was detected in the early proximal tubule and a net absorptive flux was detected along the loop of Henle, presumably the pars recta. No net flux occurred along the distal tubule. These findings are similar to those previously reported by us for non-DM rats. At the end of the clearance experiments the concentration of netilmicin in renal cortex of DM rats (56 +/- 5 micrograms/g wet wt.) was significantly less (P less than .01) than that in the renal cortex of non-DM rats (122 +/- 6 micrograms/g wet wt).(ABSTRACT TRUNCATED AT 250 WORDS)

Single-dose accumulation pharmacokinetics of tobramycin and netilmicin in normal volunteers.

The two-compartment tissue accumulation pharmacokinetics of tobramycin and netilmicin were compared in 11 normal volunteers by using a crossover design. After each 1.0-mg/kg (body weight) dose, serum was collected for 96 h, and complete 24-h urine collections were obtained for a total of 30 days. Two months of washout were required before crossover. Concentrations in serum and urine were measured by radioimmunoassay, and concentrations in serum and urinary excretion rates were simultaneously fitted to a two-compartment pharmacokinetic model. Netilmicin exhibited significantly lower total body clearance (48 versus 90 ml/min) and longer terminal elimination half-life (161 versus 96 h) than tobramycin. As a result of these pharmacokinetic differences, the predicted tissue accumulation of netilmicin at steady state was significantly higher than that of tobramycin (P less than 0.05). Relative rates of aminoglycoside nephrotoxicity probably depend on both the differential tissue uptake (accumulation) and the concentration of the aminoglycoside which produces intracellular toxicity. Because the steady-state tissue accumulation of netilmicin is nearly 2.5 times greater than that of tobramycin, its potency in the production of intracellular toxicity needs to be that much less for the two agents to produce the same incidence of clinical nephrotoxicity.

Avaliaçäo da concentraçäo inibitória mínima da ceftriaxona e de aminoglicosídeos em enterobactérias em condiçöes aeróbicas e anaeróbicas / Evaluation of minimal inhibitory concentration of ceftriaxone and aminoglycosides against enterobacteria under aerobic and anaerobic conditions

Alguns trabalhos têm demonstrado que os aminoglicosídeos apresentam uma reduçäo in vitro de sua atividade frente às enterobactérias e ao S. aureus em meios anaeróbicos. O objetivo do estudo foi o de comprovar a atividade da ceftriaxona, netilmicina e gentamicina em 30 cepas de enterobactérias isoladas de infecçöes intra e extra-hospitalares, em condiçöes de anaerobiose e aerobiose. O método para a determinaçäo da MIC foi o da diluiçäo. Os resultados demonstram que a netilmicina e gentamicina perdem (em algumas cepas) a sua atividade em condiçöes de anaerobiose, o mesmo näo ocorrendo com a ceftriaxona. Esses achados sugerem que a ceftriaxona deve ser o antibiótico de eleiçäo, nas infecçöes causadas por enterobactérias onde se suspeite de baixos níveis de oxigênio

Characteristics of gentamicin uptake in the isolated crista ampullaris of the inner ear of the guinea pig.

The characteristics of aminoglycoside uptake in the inner ear were investigated in the isolated crista ampullaris. The organ was incubated with radiolabeled gentamicin, and the stable, nonexchangeable radioactivity was considered the intracellular pool of the drug. Gentamicin was transported against a concentration gradient, and the resulting tissue to medium ratios ranged from 1.4 to 18.6. Transport was inhibited by reduction in temperature or by the addition of metabolic blockers, indicative of an energy-dependent component. The uptake system showed at least two sites, the first a high-affinity site with a dissociation constant KD = 39 nM and a capacity of n = 0.2 pmole/crista; the second had a KD = 16 microM and a capacity of n = 11 pmoles/crista. Aminoglycosides competed with uptake of gentamicin in the order netilmicin greater than or equal to neomycin greater than tobramycin, and polyamines competed in the order spermine greater than spermidine greater than putrescine; glucosamine and the basic amino acids lysine and asparagine were ineffective, excluding the participation of glucose and basic amino acid transport systems in gentamicin uptake. These results along with direct comparisons with some characteristics of putrescine uptake suggest that aminoglycoside and polyamine transport have common features in the crista ampullaris.

Aminoglycoside redistribution phenomenon after hemodialysis: netilmicin and tobramycin.

The serum concentration time profile of netilmicin and tobramycin before, during, and after hemodialysis was assessed in 5 noninfected adult chronic hemodialysis patients. The pharmacokinetic profile was biexponential for both agents in the pre-hemodialysis period. The total body clearance of netilmicin was significantly greater than that of tobramycin (5.32 +/- 0.75 ml/min vs 3.66 +/- 1.00 ml/min; p less than 0.05). The hemodialysis clearances of netilmicin and tobramycin were similar (60.8 +/- 16.6 ml/min and 54.7 +/- 18.8 ml/min, respectively). Netilmicin and tobramycin serum concentrations increased significantly 10 minutes after cessation of hemodialysis and maximally rebounded to 38.3 +/- 16.2% and 18.3 +/- 3.0% at 1.7 +/- 0.3 hours and 1.9 +/- 0.7 hours, respectively. This phenomenon may be a primary contributor to the marked variability observed in the clinical pharmacokinetics of these agents in hemodialysis patients. These data suggest that clinical serum concentrations should not be drawn until two hours after hemodialysis.

Netilmicin pharmacokinetics in uremic patients undergoing hemodialysis.

The pharmacokinetics of netilmicin after i.v. administration were studied in 10 adult hemodialyzed patients during and after a dialysis session. The mean interdialysis half-life was 49.6 h, whereas during dialysis this value was reduced to 5.02 h. The mean volume of distribution of netilmicin was about 20% of the total body weight. The dialyzer clearance of netilmicin, measured at 60 and 150 min after the beginning of the session, was about 50 ml/min; this means that 60-65% of the drug may be lost during the 4.5 h standard dialysis. The total body clearance of netilmicin was similar to the dialyzer clearance values, suggesting that the drug is eliminated almost entirely by hemodialysis and that its renal elimination in our patients is negligible. In conclusion, in uremic hemodialyzed patients netilmicin behaves like other aminoglycosides.