[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.

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 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.

Netilmicin and gentamicin multidose kinetics in normal subjects.

Multidose netilmicin and gentamicin kinetics were studied in 20 healthy subjects who received 1.7 mg/kg gentamicin (n = 10) or netilmicin (n = 10) as a 20-min infusion every 8 hr for 10 days (28 doses) during a randomized, double-blind comparative trial designed to study adverse effects and kinetics of netilmicin and gentamicin. Multidose kinetics were of the same order for gentamicin and netilmicin with the exception of the terminal plasma t 1/2 (94 and 156 hr) and the volume of distribution at steady state (450 and 1072 ml/kg). Mean peak plasma concentrations of netilmicin were slightly lower than gentamicin. Percentage of the dose eliminated in urine did not differ for the two aminoglycosides. Aminoglycoside was detectable in plasma and continued to be eliminated in urine for at least 6 days after the final dose. The plasma concentration-time profiles for both netilmicin and gentamicin were well fitted with the sum of three exponential terms; urinary excretion rates-time plots showed biexponential decay. None of the subjects had any auditory, vestibular, or renal toxicity. Although the data confirm a deep tissue compartment, they suggest that the kinetic processes involved may be more complex than previously supposed.

Pharmacokinetics of netilmicin in renal insufficiency and haemodialysis.

The pharmacokinetics of intravenously administered netilmicin, an investigational aminoglycoside antibiotic, were studied in 38 patients with creatinine clearance ranging from 150 to Oml/min/1.73m2 in order to determine the influence of kidney function status on the disposition of the antibiotic. The serum disappearance of netilmicin followed first order kinetics and the elimination rate constant decreased proportionally with decreasing renal function. Half-lives averaged 2.2 hours in normal individuals (creatinine clearance greater than 80ml/min/1.73m2) and reached 42 +/- 10 hours (mean +/- SD) in virtually anephric patients. The elimination rate constant lowered proportionally with decreasing renal function. Several linear relationships between pharmacokinetic parameters and renal function indicators were defined. A clinically useful correlation indicates that the half-life may be approximated as 3 times the serum creatinine concentration and may be used for adjustment of dosage of netilmicin in the treatment of patients with impaired renal function. During haemodialysis, netilmicin extraction from the blood reaches 75 +/- 14% (mean +-/ 95% confidence interval) of that of creatinine and 88 +/- 19% of that of blood urea nitrogen.

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.

Gentamicin, tobramycin, amikacin, and netilmicin levels in tears following intravenous administration.

Peak and trough tear and serum concentrations were determined in 27 human volunteers undergoing intravenous (IV) gentamicin sulfate, tobramycin sulfate, amikacin sulfate, and netilmicin sulfate therapy. Although effective serum concentrations were achieved, tear levels were subtherapeutic. The mean peak tear concentrations were 0.4 microgram/mL, 0.5 microgram/mL, 1.7 micrograms/mL, and 0.3 microgram/mL for gentamicin, tobramycin, amikacin, and netilmicin, respectively. These levels did not approach the minimum inhibitory concentrations for Pseudomonas and raise some concern regarding the risk-benefit ratio of IV antibiotics for bacterial keratitis.

Netilmicin sulfate: a comparative evaluation of antimicrobial activity, pharmacokinetics, adverse reactions and clinical efficacy.

Netilmicin, the 1-N-ethyl derivative of sisomicin, is a new aminoglycoside antibiotic that was recently marketed in the United States. Its role in therapeutics is not yet established. The pharmacokinetic profile of netilmicin is very similar to that of gentamicin. Its antimicrobial spectrum and clinical efficacy is similar to that of gentamicin, tobramycin and amikacin. It is less active in vitro against Pseudomonas aeruginosa that gentamicin and tobramycin, but in clinical trials the efficacy of netilmicin against the organism has been similar to other aminoglycosides. Netilmicin is active against some gentamicin and tobramycin-resistant strains of gram-negative bacilli, particularly those harboring adenylating and phosphorylation enzymes. Most of these strains are sensitive to amikacin as well, and amikacin is also active against most netilmicin-resistant strains of these bacteria. Therefore, amikacin remains the aminoglycoside of choice against gentamicin tobramycin and netilmicin-resistant gram-negative bacilli. In comparison to other currently available aminoglycosides, a lower frequency of nephrotoxicity and ototoxicity has been observed in laboratory animals given netilmicin. This has not been unequivocally demonstrated in humans. The frequency of nephrotoxicity in humans has been similar to that of other aminoglycosides. The frequency of ototoxicity associated with netilmicin in humans has been low but not significantly less than in other aminoglycosides, except in one trial. If further studies document a significantly lower frequency of ototoxicity with netilmicin, it may become the aminoglycoside of choice for patients with significant risk factors for ototoxicity, such as advanced age, renal impairment, concomitant ototoxic drug therapy and prolonged aminoglycoside administration.

Pharmacokinetics of netilmicin in hypertonic hemodiafiltration and standard hemodialysis.

Recently, we developed a peculiar model of hemodiafiltration (HDF), in which a conventional acetate hemodialysis (HD) is combined with a high flux dialyzer, a high ultrafiltration flow rate and a postdilution hypertonic reinfusion (H HDF). The pharmacokinetics of netilmicin (N), a relatively new aminoglycoside, were evaluated during 5 sessions of H HDF of 180 min and 2 sessions of HD of 270 min in the same 8 patients with a comparable blood (approximately 400 ml/min) and dialysate flow rate (approximately 520 ml/min). Additional studies were performed in 7 out of the 8 patients after 2 sessions of H HDF and one session of HD. N clearance, calculated both as plasma water and total body clearance, was so exceedingly higher during H HDF than during HD, that the amount of drug removed by H HDF in 180 min was still significantly higher than that removed by HD in 270 min. Consequently, the N half-life during HD was about 5 h, whereas during H HDF it was less than 2.5 h, approaching that reported in normal subjects. N half-life out of dialysis treatments was about 55 h. In conclusion, N pharmacokinetics are strikingly different between H HDF and HD, with N clearance during H HDF about the double of that during HD. The implications of this study are: a different dosage adjustment of aminoglycosides is needed for patients routinely treated by HDF; HDF may be a very effective treatment for the overdose of many drugs.

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.

Penetration of netilmicin into heart valves, subcutaneous and muscular tissue of patients undergoing heart surgery.

In 57 patients undergoing heart surgery concentrations of netilmicin in plasma, heart valves, muscle, and subcutaneous tissue were determined after a 5 min intravenous bolus injection of 1.5 mg/kg body weight. Within 8 h netilmicin serum concentration declined from 3 micrograms/ml to 1 microgram/ml. In heart valves the concentrations during heart surgery were high enough to inhibit most staphylococci, Klebisiella, Enterobacter and Escherichia coli strains. No different serum and tissue concentrations in patients with and without extracorporal circulation could be found.

Long-term intermittent netilmicin therapy of chronic pyelonephritis: a pharmacokinetic and clinical study.

A trial was conducted with long-term intermittent netilmicin therapy in six patients suffering from chronic pyelonephritis. Netilmicin was given in full dose for a period of 3-10 days (2-6 mg/kg/day), followed by 2 mg/kg doses once or twice weekly for 3-6 months. Individual doses were determined by computer based on the age, sex, lean body weight and serum creatinine concentrations of the patients. During the full-dose period of treatment netilmicin concentrations in serum were between 2 and 16 mg/l in serum and between 50 and 200 mg/l in urine. During intermittent treatment serum levels remained below 2 mg/l (except for 8-12 hours after dosing); in the urine concentrations were permanently in therapeutic ranges (150-4 mg/l). Renal tissue levels were simulated. Six to 12 months after long-term netilmicin treatment all patients are abacteriuric and free from symptoms. No auditory or renal toxic effects occurred.

Ototoxicity of netilmicin.

Ototoxicity and efficacy of netilmicin was evaluated in 76 consecutive patients with moderate to severe infections with Gram-negative rods and Staphylococcus aureus. Netilmicin alone was efficacious in 47 patients and in another 23 patients cure or improvement was seen with netilmicin combined with another antibiotic, usually penicillin. Therapeutic failure was seen in 4 cases, 2 of which were regarded as netilmicin failures. Vestibular function and hearing acuity was thoroughly examined by electronystagmography and audiometry. In spite of the extensive examination of the audiovestibular function it was possible to detect only one probable case of ototoxicity related to netilmicin, a reversible vestibular dysfunction. The importance of pre-therapeutic audiovestibular examination is stressed.

Intraocular penetration of netilmicin.

The intraocular penetration of the aminoglycoside netilmicin following intramuscular and subconjunctival injection was studied in 102 patients undergoing elective cataract surgery. Those who received either a single 1.5-mg/kg intramuscular injection or two 1.5-mg/kg injections 6 hours apart subsequently had therapeutic serum levels but aqueous levels of less than 2.4 micrograms/mL. Those who received a subconjunctival injection of either 12.5 or 25 mg of netilmicin subsequently had aqueous levels of up to 36 or 85.6 micrograms/mL; therapeutic levels in the anterior chamber persisted for 7 to 9 hours, with higher levels following the 25-mg injection. Despite wide variation in the aqueous levels following subconjunctival injection the ocular penetration of netilmicin was comparable to that reported for other aminoglycosides. No major complications were associated with the use of this antibiotic by either route.

[Intraocular penetration of netilmicin]. / Intraokulare Penetration von Netilmicin.

The penetration of netilmicin, a semisynthetic aminoglycosid into the primary human aqueous humor was determined. The antibacterial spectrum of netilmicin is adequate to gentamicin. The efficacy also covers gentamicin resistant strains. After intramuscular injection of 5 mg/kg body weight aqueous levels higher than the minimum inhibitory concentration (MIC) of staphylococcus aureus, Escherichia coli, Klebsiella, Enterobacter, Citrobacter and Proteus morganii were obtained. In the therapy of bacterial endophthalmitis the synergistic effect of netilmicin with beta-Lactam antibiotics in the form of a combined therapy should be made use of. For perioperative prophylaxis the newer aminoglycosids are not indicated because there is the danger of a development of resistant strains.

[Penetration of netilmicin into respiratory secretions]. / Pénétration de la nétilmicine dans les sécrétions respiratoires.

Aminoglycosides are often used in the treatment of severe Gram-negative infections, particularly those involving the respiratory tract. The purpose of this study was to evaluate the penetration of netilmicin into bronchial secretions. In 8 tracheostomized patients samples of bronchial secretions were taken at intervals through the tracheostomy cannula after intramuscular injection of netilmicin 2 mg/kg bodyweight. Concentrations of the drug were measured in bronchial secretions and in blood samples taken simultaneously, using the agar diffusion method (Grove and Randall procedure). The results showed significant penetration of netilmicin, with a mean bronchial peak reaching 3.4 micrograms/ml 1 h after the injection. Elimination was slow, with a mean residual level of 2 micrograms/ml at 6 hours. The bronchial to serum levels ratio was high (greater than 30% at 1 hour). Individual variations in both serum and bronchial levels were noted; they were unrelated to the underlying pathology. However, changes in bronchial concentrations correlated with changes in serum concentrations, which suggests passive diffusion across the blood-bronchoalveolar barrier. The fluctuations in bronchial levels and the usually low bronchial concentrations of aminoglycosides previously reported are discussed in relation to the methods used.

[Absorption, distribution, metabolism and excretion of netilmicin in rats (II). Accumulation study of netilmicin after repeated administration].

Repeated administration of netilmicin (NTL), a new aminoglycoside antibiotic, to rats at daily intramuscular dose of 10 or 20 mg/kg for 22 days did not affect the plasma level and the plasma half-life of the drug. The concentrations of NTL in the kidney increased markedly after repeated administration, and reached peak level after 8 and 15 doses for 10 and 20 mg/kg, respectively. Cumulative effect of NTL after repeated dose was also observed in the liver, spleen and lung, although the peak concentrations of NTL in these organs were below 1/50 of that in the kidney. Blood and tissue levels of NTL in rats were determined after a single intramuscular administration of 14C-NTL at a dose of 20 mg/kg following 21 repeated intramuscular administrations of NTL at daily dose of 20 mg/kg. The repeated dose of NTL had no effect on the blood level-time curve of radioactivity. The concentration of NTL in the kidney determined by radioassay was about 1/3 of that determined by bioassay, whereas the half-lives in the tissue levels determined by these two assays were nearly identical with each other. The half-life in the lung determined by bioassay was almost identical to that determined by radioassay, whereas the former was rather shorter than the latter in the liver and spleen.