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Quantification of Antibiotic Drug Potency by a Two-Compartment Radioassay of Bacterial Growth VIPA BOONKITTICHAROEN,T JAMES C

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Quantification of Antibiotic Drug Potency by a Two-Compartment Radioassay of Bacterial Growth VIPA BOONKITTICHAROEN,T JAMES C ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1990, p. 1035-1040 Vol. 34, No. 6 0066-4804/90/061035-06$02.00/0 Copyright © 1990, American Society for Microbiology Quantification of Antibiotic Drug Potency by a Two-Compartment Radioassay of Bacterial Growth VIPA BOONKITTICHAROEN,t JAMES C. EHRHARDT,* AND PETER T. KIRCHNER Department ofRadiology and Radiation Research Laboratory, The University of Iowa, Iowa City, Iowa 52242 Received 23 June 1988/Accepted 8 March 1990 The two-compartment radioassay for microbial kinetics based on continuous measurement of the 14CO2 released by bacterial metabolism of "4C-labeled substrate offers a valuable approach to testing the potency of antimicrobial drugs. By using a previously validated radioassay with gram-positive and gram-negative bacteria, a group of protein synthesis inhibitors was evaluated for their effect on microbial growth kinetics. All tested drugs induced changes in both the slopes and intercepts of the growth curves. An exponential growth model was applied to quantify the drug effect on the processes of bacterial 14CO2 liberation and cell generation. The response was measured in terms of a generation rate constant. A linear dependence of the generation rate constant on the dose of spectinomycin was observed with Escherichia coli. Sigmoidal-shaped curves were found in the assays ofchloramphenicol and tetracycline. The implications of dose-response curves are discussed on the basis of the receptor site concept for drug action. The assay sensitivities for chloramphenicol and tetracycline were similar to those obtained by the cell counting method, but the sensitivity of the radioassay was at least 10 times greater for spectinomycin. An ideal assay of antibiotic potency is the measurement of otic interaction and demonstrated the suitability of the changes in microbial kinetics that occur in response to radioassay for further studies in this area. graded increases in drug concentration (3). The quantitative (This study was conducted by V.B. in partial fulfillment of measurement of bacterial growth kinetics yields a reliable the requirements for the Ph.D. degree in Radiation Biology endpoint, the generation rate constant, which is capable of at The University of Iowa.) providing crucial information for assay design or quality control. MATERIALS AND METHODS The two-compartment radioassay of 14CO2 derived from Antibiotics. Spectinomycin hydrochloride (potency, 671 the metabolism of "'C-labeled glucose described by Budde- p,g/mg) and tetracycline hydrochloride (potency, 990 ,ug/mg) meyer (4) is an exquisite system for the study of microbial were purchased from the U.S. Pharmacopeial Convention, growth kinetics. The system is composed of a large detector Inc., Rockville, Md. Chloramphenicol (potency, 1,000 ,ug/ vial that encases a small culture vial. This design allows mg) was supplied courtesy of Parke-Davis, Div. of Warner- continuous and cumulative measurement of the evolved Lambert Co., Morris Plains, N.J. For preparation of each 14CO2 and determination of growth kinetics without further drug solution, we strictly followed standard procedures (23, sample manipulation (4, 5). Two practical problems encoun- 24). The standard solution was sterilized by membrane tered with the use of this system, i.e., low count rates and filtration and was freshly prepared for each experiment. limited growth-supporting capacity, have been solved by our organisms. and laboratory (2). Growth models for unbiased estimation of the Test Escherichia coli ATCC 25922 Staph- ylococcus aureus ATCC 29213 were chosen for two reasons: generation rate constant have been established (2a). The (i) both organisms exhibited valid exponential growth as precision of calculated generation rate constants is typically measured by the two-compartment system (2a); and (ii) the on the order of 1%. Standard strains of bacteria that exhibit valid growth behaviors by these models have been identified. use of E. coli and S. aureus provided tests of the drug assay on both gram-negative and gram-positive bacteria. The or- (Certain strains of bacteria appear to have been inhibited by ganisms were grown in Trypticase soy agar (BBL Microbi- the assay [2a] and were excluded from this study.) These ology Systems, Cockeysville, Md.) with sheep erythrocytes accomplishments have encouraged further work to test the at room temperature. Subculture was performed once a applicability of the optimized system for drug potency mea- week. surement. 14C02 detector preparation. The 14CO2 detector prepara- The purpose of this was to investigate the use of the study tion duplicated the techniques developed previously at our radioassay system for the measurement of drug potency and institution (1). The scintillation mixture was prepared from microbial kinetics in the presence of protein synthesis inhib- the stock solution of organic scintillators, which was 10 g of itors. Bacterial curves were obtained for various growth 2,5-diphenyloxazole (PPO) and 0.125 g of 1,4-di-[2]-(5-phe- concentrations of antibiotics, and mathematical models were nyloxazolyl)benzene (POPOP) in 100 ml of toluene; Triton applied to the data to produce generation rate constants. X-100; and 2 N methanolic NaOH solution. The proportions rate constants and Analysis of the relationship of generation of the three were 6:3:2. Additional insights into the bacteria-antibi- (by volume) components drug concentrations yielded PPO was added to make the final PPO content equal to 12 times the original content. A strip of Whatman no. 42 filter paper (3 by 4 cm2; Whatman, Inc., Clifton, N.J.) was folded * Corresponding author. lengthwise into a seven-creased shape. The paper was t Present address: Faculty of Medicine, Ramathibodi Hospital, dipped in the mixture for 1 min and drained to remove excess Bangkok 4, Thailand. fluid. The treated paper was dried in a light-tight desiccator. 1035 1036 BOONKITTICHAROEN ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. MICs and assay ranges for radiometric and cell counting methods Assay range (>ig/ml) Antibiotic Test organism MIC range Radiometnc Cell counting (reference no.) Spectinomycin E. coli 7.8-500 1-4 10-30 (26) Chloramphenicol E. coli 5-20 0.4-1.6 0.38-1.51 (16, 18, 19) S. aureus 3.1-12.5 0.4-1.6 Tetracycline E. coli 5-10 0.03-0.20 0.03-0.15 (18, 19) S. aureus 0.01-100 0.003-0.020 0.015-0.037 (21) a Data were obtained from a previously published report (1). Vapors of toluene and methanol were removed periodically K1K2 [C] with a vacuum pump. kapp = ko 1 (2) Bacterial suspension. Prior to each assay, the test organ- 1 + K1K2 [C]) isms were transferred from the stock culture into 10 ml of where kapp and ko are generation rate constants in the Trypticase soy broth (TSB; BBL Microbiology Systems) and presence and absence of drug, respectively; K1 is the parti- incubated at 37°C for 18 to 24 h. Bacteria from blood agar tion coefficient of the drug in the biophase; K2 is the affinity were inoculated in TSB and incubated for 2 to 3 h to obtain constant of the drug for the receptor site on the target a log-phase culture with a density of 0.2, as measured with molecule; and [C] is the drug concentration in the medium. the McFarland barium sulfate standard. The dilutions were Equation 2 can be written as: made with glucose-free TSB to obtain an approximate con- centration of 104 cells per ml. The actual concentration was kappk determined by plate counting of viable bacteria. = 1 - aC (3) Growth assay. By using the liquid scintillation vial as the ko culture vessel, 1 ml of drug solution was added to 9 ml of when K1K2 [C] << 1, and a = KjK2. Thus, in this range, the bacterial culture (i.e., 104 cells in glucose-free TSB contain- response kapp/ko is a linear function of drug concentration. ing 5 ,uCi of [U-_4C]glucose with a specific activity of 250 For the slgmoidal-shaped curve, the receptor site model, mCi/mmol). A 1-dram (3.888-g) vial containing the 14C02 equation 2, was derived by assuming that the binding of the detector was inserted into the growth chamber. Triplicate drug to the receptor site occurred on a one-to-one basis. The samples were set up for each dose level. Each assay was model was modified to define growth inhibition resulting performed under aseptic conditions to eliminate cross-con- from the binding of an average of n molecules of the drug per tamination. The assay tube was incubated in a water bath at receptor sites. Thus, 37°C and agitated at a speed of 240 rpm. 14C activity on the detector was counted for 1 min at hourly intervals with a K2 [KlCPA kapp = ko 1 (4) liquid scintillation counter. The whole process was carried 1 + K2 [KlC]n) out under subdued lighting. Mathematical treatment of data. (i) Calculation of slope and yields a sigmoidal response, and the constant n may be intercept. The background-corrected count rate R measured called the sigmoidicity factor. For the case n = 1, this at time t was assumed to follow an exponential model (2a), as simplifies to the model of Garrett (15), equation 2. follows: Equation 4 can be linearized by a logit transformation to yield the following: EVEfNo(ek, - 1) R = k = A(ekt 1) - k (1) 1ko kapp\ log ( I = log K2(K1)' + n log C (5) where E is the counting efficiency of the "4CO2 detector, fis kapp the rate constant of "4CO2 liberation (in disintegrations per The generation rate constants at each concentration were fit minute per hour per cell), No is the number of cells per to equation 3 or 5 by a least-squares regression analysis. An inoculation, k is the generation rate constant (per hour), and alternative analysis method would be to do a nonlinear A = EfNJk.
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