METHOD FOR IN ACTIVATING AND MONITORING ANTIBIOTICS AND ASSAYING FOR AMINOGLYCOSIDES

Abstract: Monitoring levels of antibiotics and inactivating same and assaying for amino glycosides with the enzyme AAC(3)-IV (acetyl coenzyme A; aminoglycoside-3-N-acetyl transferase).

Description: METHOD FOR INACTIVATING AND MONITORING ANTIBIOTICS AND ASSAYING FOR AMINOGLYCOSIDES This invention relates to materials and a method for moni¬ toring antibiotic levels in various media and for the .-removal or inactivation of aminoglycoside antibiotics. The invention will be described with reference to the monitoring of antibiotics in serum, but it will be understood that the materials and method described have application also for monitoring the levels of antibiotics in other media such as urine, effluents, animal feed, soils, fermentation systems and the like. Another important aspect of this invention has applica¬ tion to nullification of antibiotic activity when present in rela¬ tively low concentration. This enables a mixture of enzyme with cofactor to destroy antibiotic activity so that any bacteria present will manifest itself- By way of a further new and novel application, the enzyme can be used to nullify the activity of antibiotics present before assay for other antimicrobial agents. Antibiotics are prescribed by physicians for various bac¬ terial infections. While treatment with antibiotics is effec¬ tive, such treatment is oftentimes accompanied by undesirable side effects, in that such antibiotics as gentamicin, tobramycin, and arnikacin can be both nephrotoxic and ototoxic at serum con¬ centrations only slightly higher than therapeutic levels. In many instances, it is important that the concentration of the antibiotic in serum does not exceed 10 micrograms per milliliter (yg/ml) . The technique that has been adopted to protect patients and to ensure effective antibiotic dosage has been to monitor serum levels after each injection and/or during the course of treatment. Various methods of assay have been developed in which use is made of a particular enzymic reaction specific for a given antibiotic. To the present, such enzymatic assays, including the previous colorimetric assays and the present radioactive assays are very expensive, undesirably limited in their application, and the lack of activity is such as to require considerable time for completion of an assay- It is an object of this invention to provide an enzyme characterized by such high antibiotic activity as to enable use in assays or otherwise for antimicrobial agents for the removal or inactivation of antibiotics present for sterility control an whereby any bacterial presence will be able to manifest itself and in which use can be made of the enzyme in an improved proce for monitoring antibiotic levels in serum or other media withou the need for previous treatment of the serum or media in which the antibiotic level can be determined in a fraction of the tim previously required in microbiological analyses, in which the levels of a broad range of antibiotics can be determined with a single enzyme, and in which greater accuracy in measurement o antibiotic levels can be achieved. These and other objects and advantages of this invention will hereinafter appear and for purposes of illustration, but n of limitation, the invention is illustrated by the accompanying drawings in which: Figure 1 is a chart showing the time course of reaction at different enzyme concentrations, using sisomicin as the sub¬ strate and UWBl as the enzyme; Figures 2 and 3 show a graph showing linearity of assay with increasing antibiotic concentration, using gentamicin as the antibiotic and UWBl as the enzyme; Figure 4 is the same as figure 3 using kanamycin as the antibiotic and UWBl as the enzyme; and Figure 5 is the same as figures 3 and 4 with tobramycin as the antibiotic and UWBl as the enzyme. In accordance with the practice of this invention, dis¬ covery has been made of a bacterial strain from which the enzym can be easily obtained in high yields, which is extremely activ and operates to modify antibiotics to enable rapid and accurate analysis, including assays incapable of being performed by cur¬ rent enzymes, and to provide means for the rapid inactivation of aminoglycosides. The enzyme is identified by the universal nomenclature a AAC(3)-IV meaning that it is an aminoglycoside acetyl transfer¬ ase which acetylates at the 3 position of deoxystrepta ine and it is identified as the IV type because of its substrate range. 51 c v.: This enzyme can also be identified by the nomenclature acetyl coenzyme A; aminoglycoside-3-N-acetyl transferase. The enzyme is derived from the strain hereinafter referred to as UWBl which, in turn, can be derived from a naturally occurring isolate of a Klebsiella pneumoniae strain resistant to certain aminoglycoside antibiotics and which elaborates a new 3-N-acetyl-transferase. The organism Klebsiella pneumoniae was selected on the basis of its broad antibiotic resistance. The generic material responsi¬ ble for this resistance was recognized as the 3-N-acetyl trans¬ ferase. This material . was then taken as a plas id-coded 3-N-ace- tyl transferase, given the identification JR225 and incorporated into stock E. Coli cultures; following transfer, a strain is chosen that retains aminoglycoside resistance with a minimum of ' other resistance determinants. E. Coli was chosen primarily for safety and convenience. This E. Coli strain, identified as JR225/W677, and hereinafter referred to as UWBl, has only one copy of the JR225 plasmid per cell, thus it has only nominal pro¬ duction capacity. A cloned derivative of UWBl, hereinafter referred to as UWB2, having activity many times greater than UWBl was produced from plasmid DNA isolated from UWBl by using UWBl as a source of the JR225 fragments which were again incorporated into E. Coli. The result is a multicopy plasmid strain that produces aminoglycσside-3-N-acetyl transferase at a more rapid rate. The DNA is cut with restriction endonuclease Hink III and ligated in the presence of a multicopy vector. Transformation into an E. Coli recipient and selection for aminoglycoside resistance provided strain UWB2. The cloned strain UWB2 is resistant to aminoglycosides, tetracyline and ampicillin. The enzyme AAC(3)-IV was prepared in a 2 liter shaker flask containing 500 ml. of nutrient broth which was inoculated with 5 ml. of an overnight culture of UWBl or UWB2 and incubated with shaking at 37°C for 5-8 hours. The cells were harvested by centrifugation, washed with 0.1 M Tris buffer at a pH of 7.8. Cells were resuspended in 40 ml. of the same buffer and typically disrupted by passage through a French Pressure Cell or by sonic disruption. The crude extract was centrifuged at 20,000xg for 30 minutes at 4°C and the supernatant decanted and stored as the enzyme preparation. This preparation can be used with or without further purification. The enzyme catalyzes the acetylation of substrates in the presence of acetyl coenzyme A in which the substrates are represented by gentamicin, neomycin, ribostamycin, paramomy . cin, kanamycin, tobramycin, apramycin, netilmicin, and sisomici the presence of other unrelated antibiotics (e.g. tetracycline, chloramphenicol,penicillins) has been found not to interfere wi the assay. In use, a working reagent is prepared, as by combining a buffer, such as Tris buffer at a pH of 7.8, a radioactive T / acetyl-coenzyme A, such as C acetyl-coenzyme A or other labelled acetyl-coenzyme A and the crude or purified enzyme. T reaction is initiated by adding the serum sample to a measured quantity of the working reagent. The mixture is then incubated for a short period of time, such as for 5 minutes at 20-30°C, and preferably at room temperature where reaction takes place i accordance with the following known equation: . catalyst antibiotic + radioactive acetyl coenzyme A "> radioactive acetyl antibiotic - coenzyme-A Because of the great activity of the enzyme, especially that derived from UWB2, the reaction time for transfer of the labell acetyl group to the antibiotic to form the labelled acetyl ant biotic can be reduced to a matter of a few minutes, The reacti is preferably carried out at a temperature within the range of 20-30°C, and at a pH within the range of 6-8.5 and preferably 7 A portion of the incubated sample is transferred to a phosphocellulose paper support. The paper is washed one or more times with distilled water or buffer solution to remove soluble labelled acetyl coenzyme A leaving the labelled acetyla antibiotic for counting by a conventional scintillation counter -after dryine and transfer to scintillation vials. The total time for carrying out the analysis is usually less than 30 minutes. The following example describes a typical assay procedure: Example 1 Compone p n.ts used in a serum assay 10 μl C acetyl * coenzyme A 10 μl crude enzyme derived from UWBl or UWB2 5 μl 0.5 M Tris-Cl pH 7-8 10 μl serum containing antibiotic The materials were incubated for reaction for 5 minutes at room temperature. A 25 μl samυle was removed and transferred 2 to a 1cm phosphocellulose paper (P81: Whatman). The paper was washed with water, dried and counted in a toluene-based scintil- lant. A typical assay is illustrated in figure 1, which shows the time course of reaction with 10 μl of 100 μg/ml sisomicin as substrate with UWBl derived enzyme. In the assays illustrated in figures 2 and 3, the samples of gentamicin were provided in blood serum (10 μl was used for assay) with incubation for 5 minutes at 21°C with UWBl derived enzyme. In the assay illustrated in figure 4, use was made of kana ycin as the substrate in serum with UWBl derived enzyme. The assay illustrated in figure 5 was with tobramycin as an antibiotic in serum and with UWBl derived enzyme. Examples 2 to 8 In examples 2-8 the antibiotic sisomicin in example 1 is substituted by gentamicin (example 2) , tobramycin (example 3) , neomycin (example 4) , ribostamycin (example 5) , paramomycin (example 6) , kanamycin (example 7) , and apramycin (example 8) , with either UWBl or UWB2 derived enzyme in a purified or unpuri- fied state. The enzyme and the radioactive acetyl coenzyme A can be lyophilized for storage over a considerable period of time. When reconstituted for use, the materials are mixed with the serum and adjusted with the buffer for carrying out the analysis at a pH within the range of 6-8.5. It is preferred to carry out the assay at a temperature within the range of 20-30°C although the enzyme is stable at temperatures up to 60°C, without denaturation. It will be apparent from the foregoing that the acetyl transferase derived from strains UWBl or UWB2 is a stable enzym of high activity which can be used in crude or purified form to assay aminoglycoside concentrations in a variety of solution forms without interference. The antibiotics can be assayed over a wide concentration range in which the onlylimitation is the specific activity of acetyl coenzyme A at low drug con¬ centrations of less than 5 μg/ml, and the concentration of the acetyl coenzyme A at high drug concentrations. It will also be seen that the assay reaction is linear over a wide range of dru concentrations thereby to attest to the accuracy and reliabilit of the results. The following has reference to the ramification wherein by reason of the great activity of the enzyme, antibiotics can be removed or inactivated when present in various media, especially when present in small amounts, such as of en require in sterility testing. The Sterility Testing Branch of the National Center for Antibiotics Analysis conducts sterility tests on samples of all batches of antibiotics marketed in this country. In the testin procedures, the antibiotics are diluted and then filtered throu a 0.45 μ membrane which traps any contaminating bacteria. The membrane is then washed and transferred to growth media. Because of the possibility of any residual antibiotic activity remaining on the membrane after filtration, additional treatment is employed with a view towards removal or inactivati of these residues. One method that is employed resides in the addition of enzymes which inactivate certain antibiotics. This is currently being done with the addition of penicilli ase to t to the procedure involving all penicillins and cephalosporins. It has been found, in accordance with an important con¬ cept of this invention, that aminoglycoside antibiotics can be effectively and rapidly removed or inactivated by reason of the biological activity of the enzyme AAC(3)-IV . This concept will hereinafter be illustrated by the inactivation of the antibiotic gentamicin by the acetylating enzyme of this invention. Example 9 Twenty microliters of the enzyme were added to a reaction mixture containing gentamicin and acetyl coenzyme A and incubated at 30°C. At various times, 20 microliter samples were removed and added to 1/4" disks which were placed on an agar plate seeded with E. coli sensitive to gentamicin and incubated overnight at 37°C. The following day, the diameters of the zones of inhibi¬ tion were recorded as follows: Sample Zone Diameter control 15 mm 15' 15 mm 30' 13 mm 45' 11 mm 60' 9 mm 90' 7. mm It will be seen from the above that the zone diameters of the various samples decreased as the time of enzymatic incu¬ bation increased. Example 10 This example illustrates the enzymatic inactivation of gentamicin on a Millipore Membrane. Commercial samples of gentamicin obtained from Schering- Plough, were pooled and used as the gentamicin source at 40 mg/ml, 20 ml. of the gentamicin was removed aseptically and add to a flask containing 200 ml. of sterile peptone water. This w repeated for 1 other flask. Approximately 100 ml. of sterile peptone water was passe through a 0.45 micro membrane in a sterile filtering unit. The membrane was placed on a TSA plate and labelled peptone control In another filtering unit, one of the flasks of gentamic was passed through a membrane. The membrane was washed 3 times with 100 ml. of peptone water and placed on a second TSA plate labelled as gentamicin control. The other flask of gentamicin was passed through a membr as above, washed and transferred to a separate Petri Dish. 4 m of the acetylating reaction mixture and 0.1 ml. of the enzyme AAC(3)-IV were added to the dish. The dish was incubated for 1 hour at room temperature with occasional shaking. The membra was then removed and added to a TSA plate. A 48 hour culture of B. subtilis was diluted 1:10 in saline solution. The B. subtilis was spotted with a loop onto center and periphery of the membranes. The plates were then incubated over night at 37°C. The peptone control was found to have 8 well defined spo of B. subtilis growing on the center and the periphery of the membrane. The gentamicin control had no growth on any of the areas spotted. The AAC(3)-IV plates had 18 spots of B. subtili similar to that on the peptone control plate. The foregoing results clearly indicate that the gentamicin was completely ina tivated by the enzyme AAC(3)-IV such as to permit growth of B. subtilis in amounts comparable to that of the controls. A further concept of this invention resides in the proce wherein use is made of the enzyme in the assay of biological fluids for aminoglycoside content and it is a related object of this invention to provide an inexpensive, accurate, colorimetri method for assaying biological fluids for aminoglycoside conten wherein such biological fluids are characterized by the presenc of X-SH groups. It is generally recognized that to be effective as anti¬ bacterial agents the plasma level of aminoglycosides must be maintained at above about 2 μg./ml. At concentration greater than about 10 or 12 μg./ml. these antibiotics exhibit a number of serious toxic side effects in that at such levels they can be ototoxic (auditory impairment) or nephrotoxic (renal problems prior to treatment (see Smith et al. , New England Journal of Medicine, Vol. 302, pp. 1106 - 1109 (1980)). In some rare cases these antibiotics have even been implicated in neural blockades. As a consequence, close monitoring of the serum aminoglycoside concentration is desirable so that the concentration can be generally kept within the 2 - 12 μg./ml. therapeutic range to allow for the effective treatment of bacterial infection while minimizing the possible toxic side effects. Briefly described, the preferred method of this invention comprises: (1) treating the biological fluid to render the X-SH groups present insoluble and removing the insolubilized groups, (2) adjusting the remaining solution to a pH in the range from about 7.5 to about 8.2, (3) adding acetyl-coenzyme A and a thiol detecting reagent to the pH-adjusted solution, (4) measuring the optical density of the resulting solution, (5) adding the enzyme to the solution, which enzyme is char¬ acterized by a specific activity against aminoglycosides greater than about 50 μ moles product formed/min./ g. protein, (6) meas¬ uring the optical density of the resulting solution, and (7) determining the difference in the optical density values obtained. In the foregoing method, the preferred method for inso- lubilizing the X-SH groups in the biological fluid is to treat the fluid with trichloracetic acid after which the insolubilized X-SH groups can be removed from the solution by filtration or cent ifug tion. Alternatively, the X-SH groups (proteins) can be insolubi¬ lized with other acids, e.g. perchloracetic acid, by the use of heat, e.g. boiling the fluid for a few minutes, or by other known insolubilizing methods, or various combinations of any such methods. After removal of the insolubilized X-SH groups the adjust¬ ment of the pH of the resulting solution can be readily accom¬ plished by the addition of a suitable base as is well known in the art. The thiol detecting reagent is preferably 5,5'-dithio- bis(2-nitro-benzoate) because this compound is a visible color reactant, i.e. it permits the later spectropho ometrie reading to be carried out in the visible light spectrum. Other thiol detecting reagents, such as 2,2'-dithiopyridine and 4,4'-dithi pyridine, can be used but with such reagents the spectrophoto- etric values (optical densities) must be read in the ultravio spectrum. The previously described enzyme AAC(3)-IV is preferred the process for assaying for aminoglycoside content, although desirable results can be obtained by the use in the manner des cribed of other enzymes which are characterized by a specific activity in the range of from about 50-200 μg moles of product formed/min./mg./protein. About 30 seconds after the enzyme has been added, in st (5) , the optical density of the solution is determined and the difference between the two optical densities obtained is also determined. From the differences in the optical density value the original concentration of aminoglycoside in the biological fluid can be calculated, or, if preferred, can be determined b comparison with a prepared standard curve all as is well known the art. It will be understood that changes may be made in the details of formulation and operation without departing from th spirit of the invention, especially as defined in the followin claims.

Claims: WE CLAIM : 1. A method for monitoring antibiotic levels in media comprising reacting the antibiotic with a radioactive acetyl- coenzyme A of the enzyme AAC(3)-IV in accordance with the reaction: antibiotic + radioactive acetyl coenzyme A > radioactive acetyl antibiotic + coenzyme A in which the reaction is carried out at a pH of 6-8.5, separating the unreacted radioactive coenzyme A and then counting the radioactive acetyl antibiotic. 2. The method as claimed in claim 1 in which the reaction is carried out at a pH of 7-8. 3. The method as claimed in claim 1 in which the reac¬ tion is carried out in the presence of the antibiotic acetyl transferase as catalyst. 4. The method as claimed in claim 1 in which the reac¬ tion is carried out by incubating the reaction mixture for a short time at 20-30°C. 5. The method as claimed in claim 1 in which the reac¬ tion is carried out by incubating the reaction mixture a short time at about room temperature. 6. The method as claimed in claim 1 in which the antibiotic is present as a substrate in serum. 7. A method for inactivating aminoglucoside antibiotics comprising reacting the antibiotic with the acetylated enzyme AAC(3)-IV. 8. The method as claimed in claim 7 in which the reac¬ tion is carried out at a temperature within the range of room temperature to 30°C. 9. The enzyme AAC (3) -IV. 10. The enzyme as claimed in claim 9 derived from the strain UWBl and its cloned derivative UWB2. 11. A method for assaying for aminoglycoside antibiotic biological fluids comprising: (1) treating the biological fluid to render X-SH groups present in said fluid insoluble; (2) removing the insolubilized X-SH groups; (3) adjusting the remaining solution to a pH in the range from about 7.5 to about 8.2; (4) adding acetyl-coenzyme A and a thiol . detecting reagent to said pH adjusted solution; (5) measuring the optical density of the resulting solution spectrophotometrically; (6) adding an enzyme characterized by a specific activity against a inoglycosides greater than about 50 μ moles product formed/min./ g protein (7) measuring the optical density of the resulting solution; and (8) determining the difference in the optical den¬ sity values obtained in steps (5) and (7) whereby the original concentration of the amino glycoside in the biological fluid sample can be determined. 12. The method as claimed in claim 11 wherein the biolo ical fluid is blood. 13. The method as claimed in claim 12 wherein the X-SH groups in the biological fluid are rendered insoluble by treat¬ ment with trichloracetic acid. 14. The method as claimed in claim 11 wherein the X-SH groups in the biological fluid are rendered insoluble by treat¬ ment with perchloracetic acid. 15. The method as claimed in claim 11wherein the X-SH groups in the biological fluid are rendered insoluble by heating said fluid. 16. The method as claimed in claim 13 wherein the thiol detecting reagent is 5,5'-dithio-bis(2-nitrobenzoate) and the optical density is determined in the visible light spectrum. 17. The method as claimed in claim 13 wherein the thiol detecting reagent is 2,2'-dithio pyridine or 4,4'-dithio pyri- dine and the optical density is determined in the ultraviolet spectrum. 18. The method as claimed in claim 12 wherein the enzyme added is AAC(3)-IV. 19. An assay kit for determining the aminoglycoside con¬ centration in a biological fluid comprising, in packaged combi¬ nation: (1) a container holding acetyl-coenzyme A; (2) a container holding an enzyme characterized by broad spectrum activity against aminoglycosides greater than about 50 μ moles product formed/ in./mg/proteiri; (3) a container holding a thiol detecting reagent; (4) a container holding a reagent capable of insolu- bilizing X-SH groups present in biological fluids. 20. The assay kit as claimed iα claim 19 wherein the enzyme is AAC(3)-IV. 21. The assay kit as claimed in claim 20 wherein the thiol detecting reagent is 5,5'-dithio-bis-2-nitrobenzoate. 22. The assay kit as claimed in claim 21 wherein the X-SH group insolubilizing reagent is trichloracetic acid.

Inventors: DAVIES J, HARFORD S
Applicants: WISCONSIN ALUMNI RES FOUND
Application year: 1980
Main classification: C12Q1
IPC codes: C12N9, C12Q1