New Rapid Bioassay of Gentamicin Based on Luciferase Assay of Extracellular ATP in Bacterial Cultures

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, De. 1978, p /78/14-812$2./ Copyright 1978 Amerian Soiety for Mirobiology Vol. 14, No. 6 Printed in U.S.A. New Rapid Bioassay of Gentamiin Based on Luiferase Assay of Extraellular ATP in Baterial Cultures LENNART NILSSON Department of Clinial Bateriology, Linkoping University, Regionsjukhuset, S Linkoping, Sweden Reeived for publiation 19 September 1978 Baterial ATP levels in Esherihia oli ultures were determined by luiferin/luiferase assay. Rapid effets of gentamiin on ATP levels in the ultures were studied, and a dose-dependent aumulation of extraellular ATP was demonstrated. This phenomenon was used to develop a rapid bioassay of gentamiin in linial serum speimens. The auray of the assay, expressed as oeffiient of variation over the range 16 to 1,ug/ml, varied between 1.8 and 6.5%, ompared to 2.3 to 4.7% for an agar disk diffusion assay. Serum gentamiin onentrations were determined by both methods in 122 serum speimens from 43 patients reeiving gentamiin alone or in ombination with other antibiotis, and the results were ompared (r =.942). Determination of gentamiin based on luiferase assay of extraellular ATP in baterial ultures requires only 1 pl of serum, allowing for apillary sampling, and results are available within 2 h. Effets of antimirobial agents on baterial Mo.) (2) and kindly supplied by A. Lundin and A. growth an be quantified by luiferase assay of Thore, the National Defene Institute, Sundbyberg, ATP (6). Rapid bioassays of gentamiin in linial serum speimens, based on determination of itoring reagent) is ommerially available from LKB Sweden. Suh a purified luiferase reagent (ATP mon- ATP levels in baterial ultures exposed to standard gentamiin onentrations and Produts, Bromma, Sweden. Apyrase (purified grade I) and ATP were purhased from to Sigma Chemial Co. serum Other reagents were of analytial grade. Apyrase solutions were prepared daily, onsisting of.2% apyrase speimens, have reently been developed. Harber and Assher (1) determined the total ATP made up in nutrient broth (Difo Laboratories, Detroit, Mih.) ontaining 4 mm CaSO4. level in baterial ultures, and Nilsson et al. (5) seletively determined the intraellular ATP Antibioti standards. Stok aqueous solutions of level. Both methods require extration of intraellular ATP. from gentamiin sulfate, bath GMC-42-85, at erti- 1,,ug of ative gentamiin per ml were prepared In this report the relation between intraellular and extraellular ATP levels in Eshefied poteny (625,ug/mg) (Essex, Stokholm, Sweden). From this stok solution standards of 16, 8., 4., 2., and rihia oli ultures exposed to various onentrations of gentamiin has been, studied to de- of gentamiin in nutrient broth (Difo) (ph 7.4) was 1.,ug/ml were prepared in pooled human serum. In the experiment depited in Fig. 1, a serial dilution velop a rapid bioassay of the drug based on used. determination of extraellular ATP. This new Test strain. The test strain, E. oli LU 14, is a method was evaluated on linial serum speimens and ompared with an agar disk diffusion ephalothin, hlorampheniol, loxaillin, erythromy- linial isolate resistant to ampiillin, arbeniillin, assay. in, fusidi aid, linomyin, nalidixi aid, nitrofurantoin, peniillin G, and sulfaisodimidine and suseptible to gentamiin as determined by agar diffusion MATERIALS AND METHODS and broth dilution tehniques (5). Minimal inhibitory Clinial serum speimens. One hundred and onentration (.4,ug/ml) of gentamiin was determined at a density of 18 olony-forming units (CFU) twenty-two sera from 43 patients reeiving gentamiin alone or in ombination with other antibiotis were per ml. stored for a maximum of 4 weeks at -2 C before Baterial ultures. The test strain was inubated assay. in nutrient broth (5 ml in a 25-ml Erlenmeyer flask) Analytial equipment. The luminometer used in on a rotary shaker at 37 C for 16 to 18 h, at whih the luiferase assay has been desribed earlier (3). time the ulture was essentially stationary. Tenfold Light emission was registered on an Omni Sribe reorder (Houston Instrument Corp., Austin, Tex.), and giving 5 x 17 to 1 x 1W CFU/ml were inubated in 4- dilutions in nutrient broth of the 16- to 18-h ulture for registration of maximum light intensity a digital ml polystyrene tubes at 37 C without shaking in the peak holding memory volt meter model VID (G. Gay, presene of various onentrations of gentamiin. Milan, Italy) was used. Eah experiment inluded a ontrol ulture not exposed to gentamiin. The nutrient broth was supple- Analytial reagents. Firefly luiferase was purifled from FLE-5 (Sigma Chemial Co., St. Louis, mented with 1 g of gluose per liter exept for the 312 Downloaded from on November 1, 218 by guest

2 VOL. 14, 1978 NEW RAPID BIOASSAY OF GENTAMICIN 813 experiment depited in Fig. 1. In that experiment the ulture volumes were 1 ml, whereas in the other experiments.25-ml volumes were used. Determination of intraellular ATP. Samples from the baterial ultures were inubated with the ATP-hydrolyzing enzyme apyrase to eliminate extraellular ATP (6). After the apyrase treatment, intraellular ATP was extrated with H2SO4-ethylenediaminetetraaeti aid, followed by neutralization with NaOH-ontaining tris(hydroxymethyl)aminomethane buffer and ethylenediaminetetraaeti aid (4). Luiferase reagent (.4 ml) was added to the extrated sample (1. ml), whih was plaed in the luminometer, and the light intensity was reorded. The purified luiferase reagent an be added outside the luminometer beause when it is mixed with ATP the light intensity is almost onstant for several minutes (2). Standards with known amounts of ATP and reagent blanks were assayed in eah series. All assays were performed in dupliate. Determination of intraellular plus extraellular ATP. Without preeding apyrase treatment, samples from the baterial ultures were extrated and subjeted to luiferase assay as above. Determination of extraellularatp. Luiferase reagent (.1 ml) was added diretly to baterial ultures (.25 ml) or to samples (experiment in Fig. 1) without preeding extration of ATP, and the light intensity was reorded. Calulation of assay results. Sample ATP levels were alulated by using assays of standard onentrations of ATP as a referene, and orretion for bakground luminesene was made. Interferene with the assay resulting from baterial debris and/or buffer salts present in the extrats was orreted for by using an internal standard tehnique, i.e., the addition of known onentrations of ATP (3). Determination of baterial viability. Baterial numbers were determined as CFU per militer by plating after serial dilution. Determination of gentamiin by luiferase assay of extraellular ATP. A 1-pl sample of a gentamiin standard or a linial serum speimen was applied to a paper disk (6 mm in diameter) (AB Biodisk, Solna, Sweden), dried at room temperature for 2 min and plaed in a 4-ml polystyrene tube. Eah tube was inoulated with.25 ml of a 1-fold dilution of a 16- to 18-h ulture of the test strain in prewarmed nutrient broth supplemented with 1 g of gluose per liter. The ultures were inubated at 37 C for 75 min without shaking. Luiferase reagent (.1 ml) was added diretly to eah tube of baterial ulture (.25 ml). The reorded light intensity (arbitrary units) was plotted semilogarithmially against the onentration of gentamiin. All assays were performed in dupliate. Determination of gentamiin by agar disk diffusion assay. A 16- to 18-h agar disk diffusion tehnique was performed with E. oli LU 14 as the test strain and antibioti medium 7.2 (Oxoid) (ph 7.9) as the assay medium. All assays were performed in dupliate. RESULTS The relationship between viability (CFU per milliliter) and intraellular and extraellular i I- 4.o 1 1' 1 7 i6.e ' Conentration of gentamiin (g/ml) E L 'BU._ 15 :, 1 r13 FIG. 1. Relations between viability and intraellular and extraellular ATP levels in ultures of E. oli LU 14 exposed to a serial dilution ofgentamiin. After 75 min of inubation, CFU (A) and intraellular t), intraellular plus extraellular ), and extraellular ATP levels were determined. Arrow indiates the minimum inhibitory onentration of the drug against the strain. ATP levels in E. oli LU 14 ultures exposed to a serial dilution of gentamiin for 9 min is shown in Fig. 1. Viability dereased drastially at onentrations losely orresponding to the minimum inhibitory onentration of the drug against the test strain, while CFU in a ontrol ulture not exposed to gentamiin was unhanged (not shown in the figure). The aumulation of extraellular ATP ourred onomitantly with dereasing levels of intraellular ATP at gentamiin levels less than the minimum inhibitory onentration. The strongest derease of intraellular ATP levels was found at gentamiin levels losely orresponding to the miniimum inhibitory onentration, whereas aumulation of extraellular ATP was most pronouned at onentrations higher than the minimum inhibitory onentration. The amount of extraellular ATP in the ultures losely orresponded to the differene between the total ATP levels (determined after extra- Downloaded from on November 1, 218 by guest

3 814 NILSSON tion) and the intraellular ATP levels (determined after apyrase treatment and extration). The rapid dose-dependent effet of gentamiin on the aumulation of extraellular ATP made it possible to detet differenes in ATP levels within 75 min of inubation. During this time no growth (registered as CFU) was demonstrable in the unexposed ontrol ulture. The light emission reorded when luiferase reagent was added diretly to the baterial ultures exposed to standard gentamiin onentrations is shown in Fig. 2. In the range 16 to 1,ug/ml the dose-dependent aumulation of extraellular ATP was linear. Ten different experiments were statistially analyzed by the least-squares method and gave orrelation oeffiients (r) between.99 and.998 (Fig. 3). Four onentrations of gentamiin in pooled human serum were analyzed with luiferase assay of extraellular ATP and with an agar disk diffusion assay to determine the auray of these methods. For the luiferase method the oeffiient of variation and the mean reovery varied between 1.8 to 6.5% and 95 to 98%, respetively; orresponding figures for the agar disk diffusion assay were 2.3 to 4.7% and 96 to 17% (Table 1). LO). 6. S C._.C Jm t 15IO 1 5t I 'I Conentration of gentamiin (pg/mi) FIG. 2. Light emission obtained when mixing purified luiferase reagent and ultures ofe. oli LU 14 exposed for 75 min to gentamiin (16, 8, 4, 2, and 1 pg/ml) in pooled human serum. Chart speed:.2 m/min w 15 -J.S 1- -i 5S ANTIMICROB. AGENTS CHEMOTHER. DISCUSSION Rapid bioassays of gentamiin based on the determination of total (intraellular plus extraellular) ATP (1) and on intraellular ATP (5) in baterial ultures exposed to standard gentar z Conentration of gentamiin (jg/mi) FIG. 3. Ten standard graphs demonstrating the linear relation between aumulation ofextraellular ATP and log gentamiin onentrations (16 to 1 pg/ml). The level ofextraellularatp in the ultures was registered as light emission (arbitrary units). The onentrations of gentamiin in 122 linial serum speimens determined by the luiferase method and by the agar disk diffusion assay were ompared (Fig. 4). E. oli LU 14 was resistant to the antibiotis that appeared with gentamiin (5). The enountered ombinations with gentamiin were peniillins (46%), lindamyin (42%), and other antibiotis (12%). Sine the lower limit of detetion was set to 1 jig/ml, speimens yielding less, with one or both methods, were exluded from the statistial evaluation. The orrelation oeffiient between the two assays was.942, and the slope of the urve was 1.1. When sera ontaining gentamiin in presene of one or more antibiotis were analyzed separately, the orrelation oeffiient was.944 and the slope of the urve was 1.8. Downloaded from on November 1, 218 by guest

4 VOL. 14, 1978 TABLE 1. NEW RAPID BIOASSAY OF GENTAMICIN 815 Auray of determination ofgentamiin by the luiferase method and by the agar disk diffusion assay * Set-up value Deemination Coeffiient of Assay Amsay method; methodna n ~~~~~~~~~variation(% Mean (plg/ml) (pg/ml, mean ± SDb) reovery %)M Luiferase method ± ± ± ± Agar disk diffusion assay ± ± ± ± AU assays were performed in dupliate. b SD, Standard deviation. 12 letively determined by simply adding luiferase reagent to the ultures of E. oli LU 14. The determined amount losely orresponds to that 1 Y-.1x-O 3 obtained when intraellular ATP (determined rso.942 after apyrase treatment and extration) is subtrated from the total ATP (determined after 8 extration) (Fig. 1). This indiates that, in the ultures not subjeted to extration, only extraellular ATP reats with the luiferase reagent, whih presumably does not enter the baterial ell. This was further supported by luiferase * assay of ulture filtrates and of supernatants of entrifugated ultures (results not presented). 2 In ontrast to the brief flash obtained when mixing ATP and luiferase reagent, under appropriate onditions the intensity of the light emission is essentially onstant for several min utes (2). Thus, the purified luiferase reagent Agar dis diffussion assay (pg/ml) allows for addition outside the luminometer, and FIG. 4. Comparison between detern inatio.ns Of the light intensity obtained is stable during the gentamiin in linial serum speimens by luiferase assay- Sera from The linear relationship between aumulation assay and by agar disk diffusion assajy. patients reeiving gentamiin alone () ombination with other antibiotis ^) ((n = 39) were of extraellular ATP and log gentamiin onenassayed. Sera yielding less than I pg/mi 1 with ene trations in the range 16 to 1,ug/ml ould be used both methods have been exluded (n = 249). The linear as a alibration urve for the determination of regression line for sera ontaining gewntamiin in gentamiin in linial serum speimens. It was ombination with other antibiotis () w)as y = 1.8 x thus possible to develop a rapid and faile bioas say that does not require the sampling and ex- -.17, and the orrelation oeffiient wias tration steps inluded in the methods based on miin onentrations and to serum speimens luiferase assay of total (1) or intraellular ATP have reently been presented. Botth methods (5). The simpliity of the new method is evident involve extration of intraellular iktp before from the shemati outline in Fig. 5. luiferase assay. High levels of gluose in some of the linial The present study demonstrates t,he possibil- serum speimens affeted the results obtained ity of deteting dose-dependent eff ets of gen- with the luiferase method. This problem was tamiin on the aumulation of extraellular managed by the addition of 1 g of gluose per ATP in baterial ultures within 75; min of in- liter to the growth medium. Sine ombined ubation. However, the absene off adenosine antimirobial therapy is ommon, a test strain triphosphatase ativity of a bateria1 strain is a with a suitable suseptibility pattem was used. ondition for the aumulation of extraellular Determination of gentamiin with E. oli LU 14 ATP in a ulture. E. oli LU 14 setems to lak as the test strain was unaffeted by the presene suh an ativity. Extraellular ATP an be se- of other antimirobial agents in the serum spe- Downloaded from on November 1, 218 by guest

5 816 NILSSON Sa~~~~~~~mpling Culture Culture of the of the test strain test strain Clinial Speimen or Clinial nub;tion Standard Speimenl e9 Standard g Inubation S 75' Apyrase Inubat ion I 1' I Assa LfrnExtration H54ET Luisferase Neutralization a 3 Assay ifferi-n FIG. 5. Shemati outline of gentamiin deterrnination based on luiferase assay of extraellular and intraellular ATP. ANTIMICROB. AGENTS CHEMOTHER. imens. The presented results indiate that the luiferase method has a level of auray similar to the agar disk diffusion assay. The required serum sample is only 1 p1, allowing for apillary sampling. The luiferase assay makes the result available within 2 h and advie on the next dose an be given on the same day as the serum sample is reeived. Most diagnosti mirobiologial laboratories use an agar diffusion bioassay to determine the antibioti level in linial speimens. Radioimmuno- and radioenzymati assays appear to be the most aurate and reliable methods desribed, but they involve separation steps and require handling of radiohemials. The easy-to-perform, rapid, speifi, and aurate luiferase method may therefore be an attrative alternative to the established methods for determining gentamiin in linial speimens. ACKNOWLEDGMENTS This work was supported by the Swedish Board for Tehnial Development. The skillful tehnial assistane of Maud Karlsson and Stig Granstrom is gratefully aknowledged. LITERATURE CIMD 1. Harber, M. J., and A. W. Assher A new method for antibioti assay based on measurement of baterial adenosine triphosphate using the firefly bioluminesene system. J. Antimirob. Chemother. 3: Lundin, A., A. Rikardseon, and A. Thore Continuous monitoring of ATP-onverting reations by purified firefly luiferase. Anal. Biohem. 75: Lundin, A., and A. Thore Analytial information obtained by evaluation of the timeourse of the firefly bioluminesene in the assay of ATP. Anal. Biohem. 66: Lundin, A., and A. Thore Comparison of methods for extration of baterial adenine nuleotides determined by firefly assay. Appl. Mirobiol. 3: Nilsson, L., H. Hojer, A. Ansehn, and A. Thore A rapid semi-automated bioassay of gentamiin based on luiferase assay of baterial adenosine triphosphate. Sand. J. Infet. Dis. 9: Thore, A., L. Nilsson, H. Hojer, S. Ansehn, and L. Brote Effets of ampiillin on intraellular levels of adenosine triphosphate in baterial ultures related to antibioti suseptibility. Ata Pathol. Mirobiol. Sand. Set. B 85: Downloaded from on November 1, 218 by guest