Renal handling and acute urinary electrolyte effects of aminoglycoside antibiotics: Use of a solitary renal autotransplant in the conscious sheep

Renal handling of the aminoglycoside antibiotics gentamicin and tobramycin were studied before and after one hour of constant intravenous infusions adjusted to maintain a concentration of 15 μg/mL. A solitary renal autotransplant model in four conscious volume replete 40 Kg sheep was used. This unique surgical preparation allows sampling of renal arterial and renal venous blood as well as urine drained through an exteriorized parotid-ureteral fistula. This surgical preparation has considerable potential in renal pharmacology since it uses a conscious, large animal. Baseline studies in this preparation demonstrated normal, ⁵¹CrEDTA and ¹²⁵I PAH, clearances which were unaffected by the drugs. Aminoglycoside binding to pooled sheep sera was 11% at physiologic PH. calcium and magnesium concentrations. A–V difference was 1.3 ± .3 μg/mL and extraction by the kidney was 9 ± 3.2% with no differences between gentamicin and tobramycin. Clearance of gentamicin was 84% and tobramycin 86% of GFR. There was no evidence of tubular injury as evidenced by unchanged urinary beta-2 microglobulin excretion. Serum Na, K, Ca and Mg did not change over the course of the study. Both drugs caused a prompt decrease in absolute and fractional sodium excretion while only gentamicin produced a kaliuresis. Early aminoglycoside effects on electrolyte balance may be an eventual determinant of nephrotoxic potential rather than differences in renal drug handling.Nephrotoxicity is a major side effect of aminoglycoside antibiotic therapy. Although gentamicin and tobramycin have similar pharmacokinetics, including renal cortical accumulation, both double blind clinical studies (1) and experimental data (2) have shown that gentamicin is more frequently associated with renal dysfunction. Recent studies in the dog have suggested that hypokalemia due to renal potassium wasting is a risk factor predisposing to nephrotoxicity (3). In clinical usage aminoglycosides may induce hypokalemia and hypocalcemia, perhaps resulting from drug-induced magnesium depletion (4). Previous studies reporting data concerning the acute effects of aminoglycosides on renal function and electrolyte excretion have used anesthetized animals (5) or isolated perfused kidney preparations (6). The present experiments utilize a unique surgical preparation in which a solitary kidney is autotransplanted to the neck of a sheep followed by a contral ateral nephrectomy. Urine flow is exteriorized through a uretero-parotid-cutaneous fistula thus providing a conscious animal with ready access to renal arterial and renal venous blood. Our results show that renal handling of gentamicin and tobramycin do not differ during short-term constant infusions. Both drugs caused a decrease in sodium excretion while gentamicin caused a larger increase in fractional and absolute potassium excretion. This raises the possibility that nephrotoxic properties of aminoglycosides may be secondary to their effects on electrolytes.     

Determining the limits of the evolutionary potential of an antibiotic resistance gene

The AAC(6') enzymes inactivate aminoglycoside antibiotics by acetylating their substrates at the 6' position. Based on functional similarity and size similarity, the AAC(6') enzymes have been considered to be members of a single family. Our phylogenetic analysis shows that the AAC(6') enzymes instead belong to three unrelated families that we now designate as [A], [B], and [C] and that aminoglycoside acetylation at the 6' position has evolved independently at least three times. AAC(6')-Iaa is a typical member of the [A] family in that it acetylates tobramycin, kanamycin, and amikacin effectively but acetylates gentamicin ineffectively. The potential of the aac(6')-Iaa gene to increase resistance to tobramycin, kanamycin, or amikacin or to acquire resistance to gentamicin was assessed by in vitro evolution. Libraries of PCR mutagenized alleles were screened for increased resistance to tobramycin, kanamycin, and amikacin, but no isolates that conferred more resistance than the wild-type gene were recovered. The library sizes were sufficient to conclude with 99.9% confidence that no single amino acid substitution or combination of two amino acid substitutions in aac(6')-Iaa is capable of increasing resistance to the antibiotics used. It is therefore very unlikely that aac(6')-Iaa of S. typhimurium LT2 has the potential to evolve increased aminoglycoside resistance in nature. The practical implications of being able to determine the evolutionary limits for other antibiotic resistance genes are discussed.     

Pharmacokinetic analysis of topical tobramycin in equine tears by automated immunoassay

ABSTRACT: BACKGROUND: Ophthalmic antibiotic therapy in large animals is often used empirically because of the lack of pharmacokinetics studies. The purpose of the study was to determine the pharmacokinetics of topical tobramycin 0.3% ophthalmic solution in the tears of normal horses using an automated immunoassay analysis. RESULTS: The mean tobramycin concentrations in the tears at 5, 10, 15, 30 minutes and 1, 2, 4, 6 hours after administration were 759 ([PLUS-MINUS SIGN]414), 489 ([PLUS-MINUS SIGN]237), 346 ([PLUS-MINUS SIGN]227), 147 ([PLUS-MINUS SIGN]264), 27.6 ([PLUS-MINUS SIGN]28.4), 14.8 ([PLUS-MINUS SIGN]66.6), 6.7 ([PLUS-MINUS SIGN]18.6), and 23.4 ([PLUS-MINUS SIGN]73.4) mg/L. Mean tobramycin concentration was maintained above the MIC90 for commonly isolated bacteria for 68.5 min. CONCLUSION: A single dose of topical tobramycin resulted in therapeutic concentrations of tobramycin in the tears for 1 h after administration. Therapeutic levels of tobramycin remained in equine tears 6 times longer than was reported in rabbit tears.     

On the interaction between heparin and some aminoglycoside antibiotics

An interaction between the aminoglycoside antibiotics and heparin wherein charge transfer complexes are formed has been investigated to determine the degree of inhibition of antibacterial function of the antibiotic in the complexed form.Minimum inhibitory concentration (MIC) values have been obtained for the action of the aminoclycoside antibiotics tobramycin, gentamicin, amikacin, kanamycin, and streptomycin, on a sensitive strain ofE. coli. Growth curves ofE. coli determined at concentrations of these antibiotics just below the MIC demonstrated significant lengthening of the lag phase relative to control growth curves generated in the absence of antibiotic. Heparin (1 U ml^–1 and 10 U ml^–1) had no effect on control growth curves; however, particularly at the higher concentration, it reduced the effect on the lag phase produced by the aminoglycoside antibiotics. Thus kanamycin, gentamicin, and tobramycin were most affected, while amikacin and streptomycin were least affected. The rank order of inhibition of antibiotic activity by interaction with heparin was in qualitative agreement with previously published figures for the degree of complexation between antibiotics and heparin.     

Isolation and culture of airway epithelial cells from chronically infected human lungs

We describe procedures for isolating and culturing airway epithelial cells from chronically infected human lungs. Experience in our laboratory demonstrated the need to balance pathogen eradication against antibiotic toxicity to epithelial cells. To provide a logical basis for antibiotic selection and dose, we systematically analyzed the cytotoxicity of antibiotics useful against typical pathogens. Alone, colistin, ciprofloxacin, doxycycline, and tobramycin were moderately toxic at concentrations close to those used in cell culture, whereas amphotericin, ceftazidime, chloramphenicol, imipenem, meropenem, piperacillin, sulfamethoxazole/trimethoprim, and vancomycin were nontoxic even at concentrations many times the antimicrobial level. Epithelial cytotoxicity of combined antibiotics was additive, with no evidence of competition or synergism. Antibiotics had little effect on initial cell attachment and did not acutely lyse cells, but inhibited subsequent growth. Interestingly, cytotoxicity decreased markedly with increasing epithelial cell density. Cystic fibrosis (CF) and non-CF epithelial cells showed no differences in sensitivity to the antibiotics tested and initial exposure to antibiotics did not affect the electrophysiologic properties of resistance or short circuit current in well-differentiated cells. Tailored combinations of antibiotics at appropriate doses killed even multidrug-resistant bacteria. Thus, epithelial cells can usually be cultured from chronically infected CF airways.     

Eradication of biofilm cells of Staphylococcus aureus with tobramycin and cephalexin.

The kinetics of growth and formation of biofilm by Staphylococcus aureus were investigated under iron-limited conditions in the chemostat. The population of planktonic cells reached 5.5 x 10(9) cells/mL 24 h after inoculation (D = 0.05 h-1) and remained constant throughout. The number of biofilm cells of S. aureus colonizing the silicone tubing increased exponentially from 6 x 10(4) to 2.7 x 10(7) cells/cm2 (6 days later) and continued to increase at a reduced rate to 2.7 x 10(8) cells/cm2 on day 13. Planktonic cells of S. aureus were susceptible to tobramycin and cephalexin. The planktonic cells could be successfully eradicated with a combination of 5 micrograms tobramycin plus 100 micrograms cephalexin per millilitre. Exposure of young biofilm cells of S. aureus to 5 micrograms tobramycin plus 100 micrograms cephalexin per millilitre resulted in a rapid loss of cell viability. The percentage of survival dropped to less than 0.0001% after exposure to these concentrations of antibiotics for 3 h. Old biofilm cells of S. aureus were found to be extremely resistant to these antibiotics. The cell viability was reduced to 0.09% after exposure to 10 micrograms tobramycin plus 100 micrograms cephalexin per millilitre. The results suggest that it is possible to eradicate S. aureus infection at the early stage with tobramycin plus cephalexin. Any delay in implementing antibiotic therapy is likely to result in the failure of the treatment. It is important to note that the concentrations of antibiotics required for the eradication of young biofilm cells must be determined for the treatment of device-associated infections.     

Polymethyl-methacrylate-sorbitol-based capsules as local drug delivery vehicles: an in vitro antibiotic elution study

A PMMA (polymethyl-methacrylate)-sorbitol-based capsule system was recently developed, and the permeability of 16 types of capsules with different wall thicknesses and sorbitol contents tested. By optimizing these two parameters, we showed that capsule permeability could be controlled. Promising preliminary data obtained using BPB (Bromophenol Blue) as diffusion marker prompted us to further investigate the antibiotic release of capsules showing the most appropriate release characteristics. PMMA-sorbitol capsules were prepared with wall thickness of 0.5 or 0.6 mm and 60 or 70 w/w% (weight percent) of sorbitol content. In vitro gentamicin, amikacin, tobramycin releases were determined by using a microbiological agar plate diffusion assay. Capsules released 70-100% of their gentamicin load, substantially superior to Septopal, and showed preferable, extended release profiles when compared with the beads. The release kinetics of amikacin and tobramycin closely resembled those of gentamicin. PMMA-sorbitol capsules have been developed and tested, which make them promising devices for local antibiotic delivery.     

Study of the activity of a new glycopeptide antibiotic eremomycin combined with tobramycin against Staphylococci in vitro

Eremomycin is an original natural antibiotic with glycopeptide structure isolated at the Institute of New Antibiotics, the USSR Academy of Medical Sciences. Activity of eremomycin alone or in combination with tobramycin was studied with using 25 clinical strains of staphylococci. 56 and 88 per cent of the strains were respectively resistant to gentamicin and kanamycin, two aminoglycoside antibiotics. All the staphylococcal strains were sensitive to eremomycin in concentrations of 0.12 to 1 microgram/ml. The MIC of tobramycin for 10 (40 per cent) sensitive strains ranged within 0.25-2 micrograms/ml. For 60 per cent of the strains the MIC was equal to or higher than 16 micrograms/ml. When eremomycin was used in combination with tobramycin the antibacterial effect with respect to 17 strains (68 per cent) increased. In 32 per cent of the strains the effect was synergistic and in 36 per cent of the strains it was additive. Indifference and antagonism were detected with respect to 7 (28 per cent) and 1 (4% per cent) strains respectively. No significant difference was shown in manifestation of the synergistic-additive nature of eremomycin and tobramycin interaction with respect to the tobramycin sensitive and resistant strains.     

Experimental study on chemotherapeutic efficacy, acute toxic action and nephrotoxicity of combinations of eremomycin with tobramycin

Chemotherapeutic efficacy of eremomycin in combination with tobramycin was investigated on a model of experimental sepsis of albino mice caused by Staphylococcus aureus cultures resistant to methicillin. Eremomycin is a novel original antibiotic of the glycopeptide structure isolated in the USSR and tobramycin is an aminoglycoside. Acute toxicity of the combination with a wide range of the dose fixed proportions was studied on mice and the nephrotoxic action of the antibiotics and their combinations administered intravenously for 5 days was studied on albino rats. The experiments showed that the chemotherapeutic effect of eremomycin in combination with tobramycin was of synergistic nature. Acute toxicity of the combined drugs mainly summed up and somewhat increased when the proportion of tobramycin and eremomycin was 1:2.4 or 1:3.6. Eremomycin had a dose-depended nephrotoxicity. Summing up of the nephrotoxic action of the drugs on their combined use was observed.     

Antibiotic susceptibility of clinical isolates of Pseudomonas aeruginosa

Three hundred and twenty two clinical isolates of Pseudomonas aeruginosa collected in Morelia, México, were analyzed for in vitro susceptibility to five antibiotics by agar dilution tests. Antibiotic resistance was shown by 50% of total isolates. Frequencies of resistance were: streptomycin, 47%; gentamicin, 13%; tobramycin, 8%; and carbenicillin, 7%; no amikacin resistance was found. The more common resistance patterns were streptomycin, gentamicin-streptomycin, and tobramycin-gentamicin-streptomycin. Resistance to either tobramycin, gentamicin or carbenicillin was found mainly in pyocin type 10 isolates. The proportion of antibiotic resistant isolates ranged from 37 to 75% in four hospitals, and amounted 24% in three clinical laboratories.     

Monoclonal antibody based enzyme-linked immunosorbent assay for aminoglycoside antibiotic kanamycin in foodstuff

Monoclonal antibodies to aminoglycoside antibiotic kanamycin (KM) were raised as a result of mice complex immunization with glutaraldehyde conjugates BSA with KM, tobramycin (TM) and gentamicin. Using antibodies an indirect competitive enzyme-linked immunosorbent assay was developed. This method allows to determine antibiotic up to 1.2 ng/ml in water solutions, milk and eggs and up to 2.5 ng/ml in honey. The recovery rate from these products spiked with KM was 83, 84 and 96% respectively. The assay of KM based on homologous and heterologous solid-phase conjugates were estimated. The cross-reactivity with TM could vary from 7 to 54%. The same indexes for of amikacin were more constant and reached 7-8%. The other aminoglycosides showed no inhibitory activity.     

Tartrazine-containing drugs

Tobramycin, an aminoglycoside antibiotic, was used to treat 52 infections due to gram-negative organisms in 51 patients. Complicated urinary tract infections, bacteremia and pyelonephritis accounted for 80% of the infections. The rate of immediate satisfactory response was 79%. During therapy with tobramycin, resistant organisms emerged in four patients--two Pseudomonas aeruginosa and two Escherichia coli strains. There were four superinfections with tobramycin-resistant Providencia sp. In four seriously ill patients the serum creatinine concentration increased 1 mg/dL or more; in three the increase was transient. No auditory toxicity was noted in the 19 patients in whom serial audiograms were made. In vitro testing of isolates from these patients showed that tobramycin and gentamicin had equal activity against Enterobacteriaceae. Tobramycin was two to four times more active against susceptible P. aeruginosa.     

Monoclonal antibody-based enzyme-linked immunosorbent assay for the aminoglycoside antibiotic kanamycin in foodstuffs

Monoclonal antibodies to the aminoglycoside antibiotic kanamycin (KM) were obtained as a result of the complex immunization of mice with glutaraldehyde conjugates of BSA with KM, tobramycin (TM), and gentamicin. With the use of these antibodies, an indirect competitive enzyme-linked immunosorbent assay was developed. This method allows for the determination of up to 1.2 ng/ml of an antibiotic in water solutions, milk, and eggs, and up to 2.5 ng/ml in honey. The recovery rate in these products spiked with KM was 83, 84, and 96%, respectively. The assays of KM based on homologous and heterologous solid-phase conjugates were estimated. Under these conditions the cross-reactivity with TM could vary from 7 to 54%. The same indexes for amikacin were more constant and reached 7–8%. The other aminoglycosides showed no inhibitory activity.     

Effect of nickel on oxygen free radical metabolism

The effect of nickel on superoxide dismutase activity (SOD), as well as on rate of hydroxydopamine oxidation, was studied in vitro since lipid peroxidation has been implicated in cell damage by nickel, whose toxicity and carcinogenicity are well established. Nickel strongly inhibits SOD activity. The degree of inhibition is directly proportion to the nickel concentration (tested range 0.066 to 0.33 microgram/mL in the reaction mixture); to the substrate concentration (tested range 0.4 x 10 4M to 1.1 x 10 4M 6-hydroxydopamine); and to reaction mixture. Autoxidation of 6-hydroxydopamine was increased by nickel concentrations higher than 15 micrograms/mL. The combination of excessive oxygen free radical production and inhibition of their elimination by inhibition of SOD activity may contribute to the nickel toxicity that has been reported in industrial accidents, as well as to the high incidence of cancer occurring in nickel workers. It may also contribute to many complications in uremic patients, in whom increased serum nickel levels were reported to be in a similar range to those inhibiting SOD.     

Combination antibiotic therapy in an outbreak of prosthetic endocarditis caused by Staphylococcus epidermidis.

The efficacy of combination antibiotics in vivo and in vitro was studied during an outbreak of prosthetic endocarditis caused by Staphylococcus epidermidis in 10 patients. The epidemic curve suggested that patients were infected at the time of their operation, with an interval from that time until diagnosis of 11 days to 20 months. The overall mortality was 50%. Four of six patients treated with gentamicin in combination with a penicillin analogue, a cephalosporin or clindamycin survived without reoperation. One of four patients survived when treated with regimens that did not include gentamicin. In vitro studies showed a median minimum inhibitory concentration for methicillin of 8.0 microgram/mL, compared with 0.1 microgram/mL for cephalothin, clindamycin and gentamicin, and a synergistic bactericidal effect between gentamicin and methicillin, cephalothin or clindamycin. These data suggest that gentamicin is a valuable component of combination antibiotic therapy in prosthetic endocarditis caused by S. epidermidis.     

Use of breakpoint combination sensitivity testing as a simple and convenient method to evaluate the combined effects of ceftazidime and tobramycin on Pseudomonas aeruginosa and Burkholderia cepacia complex isolates in vitro

An in vitro method of determining the activity of antibiotics in combination which is simple and convenient to perform and which could be used routinely in clinical microbiology laboratories is desirable. We investigated the activity, against Pseudomonas aeruginosa and Burkholderia cepacia complex clinical isolates, of ceftazidime and tobramycin in combination using a broth macrodilution sensitivity method based on breakpoint minimum inhibitory concentrations and compared the results obtained using this method with those obtained using the microtitre checkerboard method. There was good agreement in interpretation of results between the two methods for both P. aeruginosa (90%) and B. cepacia complex isolates (70%) with tobramycin and for P. aeruginosa isolates (70%) with ceftazidime. As the breakpoint combination sensitivity testing method employs only four tubes and does not require initial determination of individual antibiotic minimum inhibitory concentrations, it is simpler and more convenient for determining the activity of antibiotics in combination than the microtitre checkerboard method. The use of this method in routine microbiology laboratories to determine the activity of antibiotic combinations against clinical isolates should optimise treatment of infection by ensuring that appropriate antibiotic combinations are prescribed.     

Isolation and characteristics of polymyxin-resistant mutants of Shigella flexneri

Some characteristics of S. flexneri 2a mutants resistant to various concentrations of polymyxin M were studied. The data indicate that mutations resulting in low (50 microgram/ml) and high (300 microgram/ml) levels of the antibiotic resistance were determined by different genes. Polymyxin resistance led to changed permeability of the outer membrane with respect to detergents and some antibiotics, such as aminoglycosides, penicillins, chloramphenicol and amphotericin B but did not change sensitivity of the strains to some bacteriophages, except phage PI. Mutants resistant to 50 microgram/ml of polymyxin M preserved their ability to induce keratoconjunctivitis in guinea pigs. Part of the strains resistant to 300 microgram/ml of the antibiotic lost this property. No correlation between the polymyxin M resistance level, loss of the pathogenic properties and toxicity of the bacterial cells was found. It was confirmed that though inactivation of endotoxin by polymyxins is associated with their capacity for interaction with lipid A, this component does not participate in development of resistance to these antibiotics.     

Determination of tobramycin in soil by HPLC with ultrasonic-assisted extraction and solid-phase extraction

Pharmaceuticals residues in the environment have become a growing scientific interest worldwide. In the light of the possible harmful effects of tobramycin, a rapid and sensitive analytical method for determination of tobramycin in soil was developed. The extraction and purification methods, derivatization conditions, and chromatographic conditions in the determination of tobramycin in soil have been fully investigated. Extraction was carried out by a combination of vortex mixer and ultrasonic oscillation using acetone/water as the extraction agent. The extract was concentrated to 1 mL and passed through the C(18) SPE cartridge rinsed with water (3 mL), methanol (3 mL). The derivatization procedure was followed by the reaction of tobramycin with 4-Chloro-3,5-dinitrobenzotrifluoride at 60°C for 10 min in pH 9.0 H(3)BO(3)-Na(2)B(4)O(7) medium. The labeled tobramycin was determined by high performance liquid chromatography at 245 nm. Separation was accomplished within 15 min in gradient elution mode with trifluoroacetic acid in mobile phase as ion-pair reagent. The correlation coefficient for the method was 0.9999 in concentrations ranging from 0.10 to 100.0 μg/g. The limit of detection was 0.02 μg/g for tobramycin in soil at a signal-to-noise ratio of 3. The calculated recoveries of the proposed method were from 78.0 to 91.0% and RSDs were 3.38-9.79% in the application to the quantitative determination of tobramycin in all types of soil. The method will help to establish adequate monitoring of tobramycin residue in soil and make the contribution to environmental behavior evaluation.     

Pharmacokinetic monitoring during aminoglycoside treatment: the optimal method for individualizing the dosage of tobramycin

The results of tobramycin concentration monitoring in 33 patients with nonspecific pulmonary infections showed a marked individual variability of the antibiotic blood levels and model-independent pharmacokinetic parameters: total clearance, steady-state volume of distribution and mean residence time whose values were distributed log-normally. Adjusting of the tobramycin dosage by the individual values of the clearance (three-point method, by concentrations 1 h (C1), 3 h (C3) and 6 h (C6), after intramuscular single administration of the antibiotic and one-point method, by C3, after repeated administrations of the antibiotic) provided by the end of a 7-day course a 1.7-fold decrease in the individual ranges of the antibiotic concentration as compared to those without the dosage adjusting. Retrospective analysis revealed that reliable individual dosing of tobramycin was provided with the simplest one-point method when the only blood specimen was collected 3 hours after the injection, i.e. the time interval inversed to the elimination rate constant. According to this method individual doses Dind were calculated by the equation Dind = DpopCpop/Cind, where pop was the population value of D and C. The values of Dind estimated in such a way did not practically differ from those estimated with the more complicated two-point (by C1 and C6) and three-point methods. Application of the equation to the tobramycin "maximum" concentration C1 or the "minimum" one (toward the end of the dosing interval, C6) resulted in less accurate and unbiased estimation of Dind.     

Determination of amikacin in cerebrospinal fluid by high-performance liquid chromatography with pulsed electrochemical detection

A highly sensitive and fast reversed-phase liquid chromatographic (LC) method combined with pulsed electrochemical detection (PED) was developed for the direct quantification of the aminoglycoside antibiotic amikacin in cerebrospinal fluid (CSF). The limit of quantification obtained was 0.06 microg/ml and linearity was established over the concentration range 0.06-4.00 microg/ml. The recovery was found to be close to 100%. This method was developed in order to study CSF pharmacokinetics of amikacin in neonates. The narrow therapeutic range calls for monitoring to ensure optimal therapy and to minimize the risk of toxic side effects such as nephro- and ototoxicity, especially in populations like preterm neonates at birth, where the predictability of amikacin clearance is limited. Typical problems to be solved were the low amikacin concentrations and the limited sample volume of CSF.