R IC H A R D C. T IL T O N, Ph.D. A N D L IN D A L IE B E R M A N, B.S.

Transcription

1 A n n a l s o f C l i n i c a l a n d L a b o r a t o r y S c i e n c e, Vol. 4, No. 3 Copyright 1974, Institute for Clinical Science M icrodilution Assay o f Antibiotics in Body Flu ids R IC H A R D C. T IL T O N, Ph.D. A N D L IN D A L IE B E R M A N, B.S. Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington, CT ABSTRACT A method is described for the microdilution assay of antibiotics in body fluids. The effect of inoculum strength on the assay was determined. Comparison of the microdilution test, disc agar diffusion assay and radioimmunoassay for gentamicin revealed satisfactory agreement among the three tests. In tro d u ctio n Many factors have influenced the increased demand for the assay of antibiotics in body fluids. Among them are the use of toxic antibiotics such as gentamicin and the appearance of bizarre opportunistic pathogens which are difficult to eradicate without knowledge of the minimum inhibitory concentration (M IC ) and the blood level of the antibiotic as well as the trend towards more precise antimicrobial therapy. The technical approach to the bioassay of antibiotics has been multi-faceted. The two standard methods, the cylinder plate technique5 and the tube dilution-turbidometric technique,9 have been supplanted by numerous modifications and automated systems.2 Smith et al10 has proposed a method for gentamicin assay which employs R-factor mediated gentamicin adenylating enzymes from Escherichia coli. Lewis et al6 and Mahon et al7 have reported a radioimmunoassay for gentamicin. Abramowicz1 described a micro tube dilution assay for penicillin in the serum of newborn infants. The present method is applicable to the routine clinical laboratory and does not require sophisticated or expensive equipment. M ethods o f P rocedure S a m p l e P r e p a r a t i o n Serum was inactivated prior to testing at 56 for 30 minutes in a serological water bath. If other body fluids such as urine or cerebrospinal fluid were thought to contain microorganisms, they were sterilized by membrane filtration (0.22 /xm) before testing. Sputum submitted for gentamicin assay was boiled for five minutes, centrifuged at 5,000 X g and the clear supernatant fluid removed for assay (gentamicin is stable at 100 ). D i l u t i o n o f t h e S a m p l e An arithmetic dilution series was made from initial sample dilutions of 1:2, 1:2.5, 1:3, and 1:3.5 in a multiwelled polystyrene tray.* After simultaneous serial twofold di * Canalco, Inc.

2 M ICRODILUTION ASSAY OF ANTIBIOTICS IN BODY FLUIDS lution of the four rows A, B, C and D, the resulting dilution series was 1:2, 2.5, 3.0, 3.5, 4, 5, 6, 7, 8, 10, etc. The detailed procedure is shown in table I. Reagents were added to the trays using 25 and 50 microliter sterile disposable dropping pipettes.f Starting with well #2, the mixtures were serially diluted with microtiter loops, f Following dilution, 50, 75, 100, and 125 microliters of inoculum were added to each well in rows A, B, C and D, respectively. The plates were sealed with cellophane tape and incubated at 35 overnight. This procedure has been automated using the Autotiter IV * at a significant time savings. T A B L E I A r i t h m e t i c D i l u t i o n S e r i e s Initial Well Ml Well #2 Well - #10 Serum Growth Growth Row Dilution Serum* Medium* Serum * Medium* Serum* A 1: B 1 : C 1 : D 1 : * M icroliters R e f e r e n c e A n t ib io t ic s Antibiotics were purchased in a set,* prepared as described elsewhere12 and stored at 20 until used. Reference solutions of antibiotics were never refrozen nor held in excess of two weeks in the freezer. I n o c u l u m The bacterial inoculum was standardized at 1 X 105 colony forming units per ml (cfu per m l) as described elsewhere.12 R e f e r e n c e B a c t e r ia l S t r a in s These bacteria were used as reference assay organisms for their respective antibiotics: Escherichia coli WHO-5, ampicillin, cephalothin; E. coli 4883, j gentamicin; Staph, aureus 2834,* methicillin. The minimum inhibitory concentration (M IC ) for each antibiotic-organism combination was determined on a minimum of 50 separate occasions11 prior to its designation as an assay organism. The organisms were maintained on trypticase soy agar slants and transferred at weekly intervals. G r o w t h M e d iu m Mueller-Hinton broth ( M H ) ( B B L ) was employed throughout the study and sterilized by autoclaving for 15 minutes at 121. f Dynatech, Inc. * Canalco, Inc. Provided by Thomas Gavan, M.D., Cleveland Clinic Foundation, Cleveland, OH. R e a d in g o f t h e T r a y s Following incubation, the trays were read on a viewer.* The highest dilution of serum showing no detectable growth was determined to be the endpoint. Growth was defined as (1) confluent turbidity or (2 ) single or multiple clusters of growth = > 2 mm diameter. Q u a l it y C o n t r o l Each assay plate included controls on sample sterility, growth medium sterility and inoculum reactivity. Inocula were plated on 10 percent sheep blood agar to insure purity. The Autotiter IV was quality controlled as described by Gerlach.4 C a l c u l a t io n s The concentration of biologically active antibiotic was calculated by multiplying the reciprocal of the highest serum dilution showing no growth times the MIC of the assay organism. For example: (1) highest dilution of serum showing no growth = 1:8; (2) MIC of assay organisms = 0.5 meg per ml and (3) antibiotic concentration = 4 meg per ml.

3 1 8 0 T IL T O N AND L IE B E B M A N T A B L E II R e c o v e r y o p A n t i b i o t i c f r o m N o r m a l a n d Antibiotic Added H e a t I n a c t i v a t e d N o r m a l S e r u m * meg/ml Experimental Heated Unheated Ampicillin Methicillin Cephalothin Gentamicin *Serial 2-fold dilution. Unheated normal serum is inhibitory at a. 1: dilution. N o b m a l S e b u m Serum for experimental recovery of antibiotics was drawn only from volunteers who had not been treated with antibiotics for a minimum of three months. R esu lts R e p r o d u c ib il it y Twenty-four consecutive cephalothin assays and 18 consecutive ampicillin assays were performed in serum by the Autotiter and by the manual microtiter method. The results for all machine-performed assays were identical while two of 24 cephalothin assays and three of 18 ampicillin assays were one dilution interval from the mean by manual methods. R e c o v e r y o f A d d e d A n t i b io t ic f r o m S e r u m Known amounts of ampicillin, cephalothin, methicillin and gentamicin were added to serum. The sera were assayed in triplicate by automated serial twofold micro dilution before and after heat inactivation. Results are shown in table II and reflect the mean of the three replicate assays. In most instances, 100 percent of the antibiotic activity was recovered. Results in table II also show that unheated serum from donors not being treated with antibiotic was inhibitory to the test bacteria at dilutions of 1:2 to 1:4. Inactivation of serum at 56 for 30 minutes removed this inhibitory effect and did not alter the recovery of added antibiotic from serum. E f f e c t o f I n o c u l u m S iz e o n t h e S e r u m A s s a y In table III are shown the results of a marked effect on apparent antibiotic concentration when the inoculum size was varied from 1 X 105 cfu per ml to 1 X 107 cfu per ml. Assays were performed in triplicate and instances where such triplicate assays were not identical are noted in the table. The effect was most marked when assaying ampicillin and methicillin with spuriously high results being noted when the inoculum size was increased. T A B L E III E f f e c t o f I n o c u l u m S t r e n g t h o n R e c o v e r y Antibiotic o f A n t i b i o t i c s f r o m N o r m a l S e r u m * Actual Antibiotic Experimental cfu/ml ( antibiotic) I05 cfu/ml 10$ cfu/ml Cephalo thin Ampi / /512+ cillin Hethi /128t /512+ cillin / Genta /512t micin / *Serial two-fold dilution. +Denotes variation, from triplicate mean.

4 MICRODILUTION ASSAY OF ANTIBIOTICS IN BODY FLUIDS TA BLE IV R e c o v e r y o f A d d e d A n t i b i o t i c s U s i n g a n A n tib io tic Added A r i t h m e t i c D i l u t i o n T e c h n i q u e Recovered A m p i c i l l i n C e p h a l o t h i n M e t h i c i l l i n G e n t a m ic in R e c o v e r y o f A n t ib io t ic s f b o m S e r u m b y A r i t h m e t i c D il u t io n The results of antibiotic recovery by the arithmetic dilution technique performed in triplicate are shown in table IV. The assay was accurate at antibiotic concentrations less than 10 micrograms per ml. At concentrations greater than 10 micrograms per ml, there were errors of from 20 to 33 percent. was significantly higher (8.4 meg per ml) than either MD (1.0 meg per m l) or RIA (0.7 meg per m l). The MD result of <1.0 meg per ml reflects the limits of sensitivity for that assay organism. D iscussion The changing patterns of antibiotic resistance of bacteria3 and the development of toxic antibiotics coupled with the increased use of quantitative antimicrobial susceptibility tests (M IC ) have led to an increased demand for antibiotic assay. The method as described offers substantial advantages over manual tube dilution assays: (1) no requirement for expensive equipment but provision for the use of automated serial dilutors (Autotiter) if desired, (2) maximum sample volume of 0.5 ml, (3) reduction of dilutional error by arithmetic dilution series, (4) use of a standardized assay microorganism and (5) minimum cost per assay. The chief disadvantage is one of incubation time (18 hours) although recent experiments using T A B L E V C o m p a r i s o n o f T h r e e M e t h o d s f o r G e n t a m i c i n A s s a y i n T e n P a t i e n t s B e i n g T r e a t e d w i t h G e n t a m i c i n C o m p a r is o n o f A s s a y M e t h o d s Blood was drawn from ten patients being treated with gentamicin for severe infections. The serum sample was divided in three parts for microdilution assay (M D ), disc agar diffusion assay (D A D ) on six patients8 and radioimmunoassay (R IA ).6 7 Results of the three assays are shown in table V. In three patients (numbers 5, 9 and 10) there was closer agreement between the MD and the DAD method than RIA. In only one patient (number 6) did the result of the MD assay fail to correspond with at least one of the other methods. In patient number 8, the DAD result P a t ie n t No. M icrod ilu t io n G en tam icin R a d io - immuno- A ssay D i s c / a g a r D iffu s io n _ < <

5 1 8 2 T ILTO N AND L IE B E R M A N a more sensitive indicator of microbial growth suggest that a one-day assay is possible. Tube dilution antibiotic assays are time consuming and suffer from an inherent problem of accuracy; that is, a one tube variation in result leads to a 100 percent error in concentration. The use of an arithmetic instead of a geometric dilution series as described reduces the theoretical error significantly. In the experiments designed to test the recovery of known amounts of antibiotic before and after heating as well as the experiment to detect variation owing to inoculum size, a single serial twofold serial dilution was used in order to maximize the error induced by these variables. However, in the comparison of the three assay methods ( table V ) and on all routine patients specimens, arithmetic dilution series were used for increased accuracy. As shown in table II, with few exceptions, the recovery of known amounts of antibiotic from serum was adequate even after heating to inactivate complement. Of the antibiotics tested, serum levels of ampicillin and methicillin rarely exceed 10 meg per ml, gentamicin in excess of 12 meg per ml is toxic and cephalothin serum concentrations usually do not exceed 20 meg per ml. Clinical experience has shown that the accuracy of the test at fluid concentrations of antibiotic less than 10 meg per ml has been adequate to detect the expected therapeutic levels. Other dilutional methods suggest the use of the patient s organism as an assay organism.9 In many situations, the organism may not be available or it may not be amenable to growth under the standard conditions of the assay. There is added work in determining a concomitant MIC for the assay organism. If a microdilution assay is used, there are advantages in using a reference assay organism whose growth requirements are known and the M IC for a particular antibiotic has been well standardized. The use of a reference organism increases accuracy and precision. Tilton et al11 have shown that variation in MIC is seen as a function of inoculum size. The same variation, but to a lesser extent, is seen with microdilution assays. An initial inoculum of 1 X 105 cfu per ml is recommended for both the MIC and the assay procedure. The comparison of the MD, RIA, and DAD assay for gentamicin indicated that, in most cases, there was agreement between MD and RIA. Where there was disagreement, in no case would the results have altered the therapeutic regime of the patient or indicated a toxic serum level of antibiotic. The benefits of RIA are time (three hours) and lack of interference by other antibiotic substances. It is possible, however, to pretreat serum with /3-lactamase to inactivate the penicillins and cephalosporins prior to assaying by either MD or DAD for /5-lactamase resistant antibiotics. It is also possible to assay antibiotics by MD in the presence of the sulfonamides by performing parallel assays in two media, one without paraaminobenzoic acid (PABA) and one with PABA. Although the MD assay was conceived originally for serum, it has been performed on urine, synovial fluid, pus and sputum after liquefaction with similar results. The assay is not restricted to the antibiotics described in this report. Any antibiotic can be assayed if there is a fast-growing bacterium available with a known MIC for that particular antibiotic. The relative ease of performance of the assay allows the construction of pharmacokinetic curves in which a baseline serum sample is drawn prior to treatment followed by serum samples at time intervals subsequent to the administration of the drug. The data indicate that the antibiotic as

6 MICRODILUTION ASSAY OF ANTIBIOTICS IN BODY FLUIDS say described can be effectively used as a routine laboratory procedure with minimal outlay for equipment. R eferences 1. A b r a m o w i c z, M., K l e i n, J. O., In g a l l, D., a n d F i n l a n d, M.: Levels of penicillin in serum of newborn infants. Amer. J. Dis. Children 111: , B u r n s, D. A. a n d H a n s e n, G. D.: Total automation of an antibiotic assay. Ann. NY Acad. Sci. 153: , F i n l a n d, M.: Changing ecology of bacterial infections as related to antibacterial therapy. J. Infect. Dis. 122:419^131, G e r l a c h, E. H.: Microdilution I: A comparative study. Current Techniques for Antibiotic Susceptibility Testing. Arthur C. Thomas, Inc., Philadelphia, G r o v e, D. C. a n d R a n d a l l, W. A.: Assay method of antibiotics: a laboratory manual. Medical Encyclopedia, Inc., New York, L e w i s, J. E., N e l s o n, J. C., a n d E l d e r, H. A.: Radioimmunoassay of an antibiotic: gentamicin. Nature New Biology 239: , M a h o n, W. A., E z e r, J., a n d W i l s o n, T. W. : Radioimmunoassay for measurement of gentamicin in blood. Antimicrob. Ag. Chemoth. 3: , S a b a t h, L. D., C a s e y, J. I., R u c h, P. A., S t u m p f, L. L., a n d F i n l a n d, M.: Rapid assay for circulating nephrotic antibiotics. Antimicro. Ag. Chemoth. 1:83-90, S c h l i c t e r, J. G., M a c L e a n, H., a n d M a l z e r, A.: Effective penicillin therapy in subacute bacterial endocarditis and other chronic infections. Amer. J. Med. Sci. 217: , S m i t h, D. H., V a n O t t o, B., a n d S m i t h, A. L. : A rapid chemical assay for gentamicin. N. Eng. J. Med. 286: , T i l t o n, R. C., L i e b e r m a n, L. B., a n d G e r l a c h, E. H.: The microdilution antibiotic susceptibility test: an examination of certain variables. Appl. Microbiol., 1973 (in press). 12. T i l t o n, R. C. a n d N e w b e r g, L. B.: Standardization of the microdilution susceptibility test. Current Techniques for Antibiotic Susceptibility Testing, Arthur C. Thomas, Inc., Philadelphia, 1973.