Aminoglycoside assays

Urease assay. When Proteus mirabilis grows in a urea-containing medium it hydrolyses the urea to ammonia and consequently raises the pH of the medium. This production of urease is inhibited by aminoglycoside antibiotics (inhibitors of protein synthesis Chapter 8). In practice, it is difficult to obtain reliable results by this method. 

Luciferase assay. In this technique, firefly luciferase is used to measure small amounts of adenosine triphosphate (ATP) in a bacterial culture, ATP levels being reduced by the inhibitory action of aminoglycoside antibiotics. This method may find more application in the future as more active and reliable luciferase preparations become available. 

White L.O. Reeves D.S. (1983) Enzymatic assay of aminoglycoside antibiotics. In Antibiotics Assessment of Antimicrobial Activity and Resistance (eds A.D. Russell L.B. Quesnel), pp. 199-210. Society for Applied Bacteriology Technical Series No. 18. London Academic Press. 

An enzymic assay involving the reaction of neomycin with aminoglycoside 4 adenyltransferase has been described l. a linear response for neomycin was observed over the range 2.5 to 20yg/ml serum. 

E. E., Risen, L., Griffey, R. H. Design and synthesis of paromomycin-related heterocycle-substituted aminoglycoside mimetics based on a mass spectrometry RNA-binding assay. 

The synthesized 3, 4 -dideoxyaminoglycosides were assayed against aminoglycoside snsceptible and resistant strains of E. coli (Table 4.7). As expected, both RR501 and RT501 are active against resistant bacteria eqnipped with APH(3 ). They are, however, less active against bacteria eqnipped with AAC(6 )/APH(2")". 

Aminoglycosides remain clinically important antibiotics. NMR provided the initial breakthrough in structural understanding of aminoglycoside action on the ribosome, and it remains a powerful tool for the biophysical characterization of drug-RNA interaction. The combined use of NMR, X-ray crystallography, thermodynamic and functional assays, and computational methods is needed to drive forward the development of new aminoglycosides with improved clinical properties. The rich data described above, combined with the application of new synthetic methods, bode well for the future. 

While the binding of aminoglycosides to the RRE provides a proof of principle, their affinity and, in particular, selectivity traits need to be improved for true therapeutic utility. To facilitate the discovery of potent and selective RRE binders, we developed a solid-phase assay. The components of this assembly include (a) insoluble agarose beads (or microtiter plates) covalently modified with streptavidin, (b) a biotinylated RRE fragment, and (c) a fluorescein-labeled Rev fragment (RevFl). Assembly of the three components generates an immobilized ternary complex whereby the biotinylated RRE binds to the beaded... 

Crystal strnctnres of a DIS construct bound to aminoglycosides have been pnblished. A flnorescence-based assay for evalnating ligand binding to DIS has been reported. 

More versatile than the growth-inhibition assays and potentially applicable to determining the presence of different antibiotic residues in different matrices are the microbial receptor CHARM I and II test assays (19, 20). The Charm I test, developed exclusively for -lactams in milk, constitutes the first rapid test recognized by The Association of Official Analytical Chemists (AOAC) with a test time of 15 min (19). The speed and sensitivity of this test permitted testing of milk tankers before they unloaded at the processing plant (21). In 1984-1985, the CHARM I test was further developed to test for antibiotics beyond -lactams to include tetracyclines, sulfonamides, aminoglycosides, chloramphenicol, novobiocin, and macrolides. The extended version has been referred to as CHARM II test. 

Clinical reports on tobramycin continued to describe cures in a variety of Gram-negative infections.1+8 A radioimmune assay was described.1 9 Initial explorations into 15N-NMR spectra of several of the nebramycin components suggested a promising tool for following aminoglycoside modification.50... 

A 5. oirautans mutant was Isolated which produced ribostamycin free of xylostasin.A rapid luciferase-based assay of gentamicin was reported Labeled sisomicin was found to be taken up into bacterial cells at a higher rate than gentamicin. The binding of aminoglycosides to acidic mucopolysaccharides was studied. ... 

The MASS assay was used to determine the Kd values for paromomycin, kanamycin A, and neamine. While the aminoglycoside anhbiotics were screened at 250 nM, 750 nM. 2.5 /trM, and 7.5 /rM, 16S was held at 100 nM. Figure 3.2 shows a plot of the frachon of 16S bound by the hgand (fraction bound) versus the total hgand concentrahon for the three aminogylcosides. The Kd values determined for paromomycin (83.9 nM), kanamycin A (7.8 I M), and neamine (13.1 /zM) are consistent with soluhon phase determina-hons [26,30] and our previous ESI-MS measurements [18]. Each Kd determinahon took 380 seconds of data acquisihon. Thus, the MASS assay provides a robust way to determine Kd values for 100 compounds a day. 

Thus the ehmination of aminoglycosides in ESKD patients also receiving antipseudomonal penichlins will be increased therefore frequent serum concentration monitoring should be performed. To minimize any in vitro inactivation of aminoglycosides that would comphcate assessment of the in vivo effects, serum samples should be assayed as soon as possible after coUection. If this is not possible, serum samples should be frozen (preferably at-70°C, or-94°F) until they can be assayed. 

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