RAPID MICROBIOLOGICAL ASSAY OF ANTIBIOTIC IN BLOOD AND OTHER BODY FLUIDS

375

Of the children in group (e) who did not produce H.G.H. in response to Bovril, two had a craniopharyngioma, three had evidence of brain damage, and one had Turner’s syndrome. Serum-insulin and blood-glucose levels were determined also in five of the children who responded to Bovril. No significant changes occurred (see fig. 2). 7% of all children refused to drink the Bovril. Comparison of Responses to Bovril and Insulin-induced Hypo-

glycaemia

responding to neither. Discussion The H.G.H. responses to Bovril in children are similar to those reported as following other stimuli such as hypoglycaemia induced by intravenous insulin (Wolter

and Loeb 1967,

Kaplan et al. 1968) or the infusion of arginine (Rosselin et al. 1967). In addition, in five of our children in whom the H.G.H. was measured following Bovril and following intravenous insulin, similar responses obtained.

Prepubertal boys and girls and women responded to Bovril, but only one of the men. Sex differences in the secretion of H.G.H. in adults have been noted previously. Ambulatory values were found to be higher in women than in men (Frantz and Rabkin 1965). Merimee et al. (1966) observed that women gave greater responses to intravenous arginine than men, and that the response of the latter could be increased by administering stilboestrol. They suggested that oestrogens sensitised the pituitary or hypothalamus to normal H.G.H.-releasing stimuli. The changes in H.G.H. levels following Bovril in a series of children with growth retardation were similar to those observed by Kaplan et al. (1968) following hypoglycxmia; good responses were obtained in small normal children, and negligible responses in children with hypopituitarism. In our series two short children with maternal deprivation failed to respond-a similar finding was recorded by Powell et al. (1967). The mechanism by which Bovril stimulates H.G.H. is unknown. The response is not dependant on changes in blood-sugar, and unlike arginine and other aminoacids (Floyd Jr. et al. 1965) it does not cause a release of insulin. Bovril is a complex mixture of substances, and investigations are in progress to try and identify the compound which results in H.G.H.-release. This test has obvious advantages in paediatrics since no intravenous infusion is required, it is completely safe, and the child requires no

special nursing

care.

We are grateful to the physicians of the Hospital for Sick Children for allowing us to investigate their patients; to Dr. S. Raiti for the samples following intravenous insulin; to Dr. D. R. Boyns for advice and a gift of antiserum prepared by Dr. D. Wright; to Mrs. S. Chalkley, B.SC., for iodinating the growth-hormone; and to Dr. F. C. Greenwood for gifts of radioactive iodine. We gratefully acknowledge financial help from the Medical Research Council. Requests for reprints should be addressed to B. C., Department of Chemical Pathology, Hospital for Sick Children, Great Ormond Street, London W.C.I. REFERENCES

B. E., Tanner, J. M., Newns, G. H., Whitehouse, R. H., Renwick, A. G. C. (1967) Archs Dis. Childh. 42, 245. Floyd, Jr., J. C., Fajans, S. S., Knopf, R. F., Rull, J., Conn, J. W. (1965) Clin. Res. 13, 322. Frantz, A. G., Rabkin, M. T. (1965) J. clin. Endocr. Metab. 25, 1470. Grant, D. B. (1967) Archs Dis. Childh. 42, 375.

Clayton,

M.D.

S. FAINE N.Z., D.Phil. Oxon., M.C.P.A.

PROFESSOR OF

MICROBIOLOGY, MONASH

UNIVERSITY

D. C. KNIGHT MEDICAL

in Children

Seven children were given both a Bovril test and an intravenous-insulin test. The results (table iv) were similar for both tests, four of the children responding to Bovril and to insulin-induced hypoglycsemia, and three

were

RAPID MICROBIOLOGICAL ASSAY OF ANTIBIOTIC IN BLOOD AND OTHER BODY FLUIDS

STUDENT, UNIVERSITY

OF SYDNEY

From the Department of Microbiology, Monash University Medical School, Alfred Hospital, Melbourne, and the Department of Bacteriology, University of Sydney, Sydney, Australia.

of antibiotic (kanamycin, have been measured in 40 gentamicin) minutes to 1½ hours with an accuracy and a reproducibility comparable with plate or tube assay methods, using samples of 0·1-0·6 ml. of serum. The principle is based on measurement of fall in pH in a culture containing a heavy inoculum of test organisms. Fermentation is inhibited by antibiotic, and the degree of inhibition reflected in the pH change is related to the concentration of antibiotic. Two methods are available, both lending themselves to feeding of instrumented data into an automated integrated laboratory-data-processing system. One involves a single measurement of pH after 1½ hours’ incubation; the other makes use of a continuous recording and depends on the rate of change of pH.

Summary

Blood-levels

Introduction

To control accurately the dosage of an antibiotic, it is necessary to know the concentration in blood. This is

particularly important in patients undergoing treatment with kanamycin or gentamicin in whom renal function is impaired. They cannot excrete the antibiotic normally, and may, therefore, build up potentially toxic bloodantibiotic levels after usual dosage schedules. Methods available at present for measuring blood-levels require overnight or 6-8 hours’ incubation in a tube-dilution or plate-diffusion blood assay. Rapid measurement of the antibiotic concentration in blood would allow accurate adjustment of frequency and amount of dose, so as to achieve the best therapeutic effect without reaching toxic blood-levels. Two new methods are described here, by which antibiotic concentration can be measured in samples of 0-1-0-6 ml. blood in under 2 hours. The principle is that small changes in pH in rapidly metabolising cultures of selected bacteria may be measured with a sensitive pH meter. Change in pH is a function of antibiotic concentration, within limits to be stated. In the first method final pH is measured after a given period of DOREEN

JACKSON

AND OTHERS:

REFERENCES—continued

Greenwood, F. C., Hunter, W. M., Glover, J. S. (1963) Biochem. J. 89, 114. Huggett, A. St. G., Nixon, D. A. (1957) Lancet, ii, 368. Kaplan, S. L., Abrams, C. A. L., Bell, J. J., Conte, F. A., Grumbach, M. M. (1968) Pediat. Res. 2, 43. Merimee, T. J., Burgess, J. A., Rabinowitz, D. (1966) J. clin. Endocr. Metab. 26, 791. Lillicrap, D. A., Rabinowitz, D. (1965) Lancet, ii, 668. Morgan, C. R., Lazarow, A. (1963) Diabetes, 12, 115. Powell, G. F., Brasel, J. A., Raiti, S., Blizzard, R. M. (1967) New Engl. J. Med. 276, 1279. Quabbe, H-J., Schilling, E., Helge, H. (1966) J. clin. Endocr. Metab. 26, 1173. Rosselin, G., Tchobroutsky, G., Assan, R., Freychet, P. (1967) Excerpta Medica, International Congress Series no. 142, abstract 82. Roth, J., Glick, S. M., Yalow, R. S., Berson, S. A. (1963) Science, N.Y. 140, 987. Wolter, R., Loeb, H. (1967) Excerpta Medica, International Congress Series no. 142, abstract 83. —

376

incubation; in the second, the during incubation.

rate

of

change of pH

is

measured

Materials and Methods Culture Klebsiella aerogenes (laboratory identification strain " Carlos ") isolated from urine from a child in 1966, and kindly provided by Dr. D. C. Dorman, was used. It was maintained on nutrient agar. Stocks were subcultured by spreading on the surface of buffered (pH 7-0) 1% peptone agar containing 1% lactose, and incubated at 37°C overnight. Growth was washed off the agar with peptone water, and the suspension was held at 4°C until use. Activity of suspensions did not change when tested repeatedly during 10 hours. The suspension density was approximately 10" per ml., equivalent to an inoculum of approximately 4 x 109 live organisms (Miles and Misra 1938).

Antibiotics

Kanamycin sulphate (crystalline, batch no. G.5194, potency mg.) kindly provided by Bristol Laboratories; gentamicin sulphate (crystalline, no. 990010, potency 595 g. per mg.), kindly provided by Schering (Australia) Ltd. Sensitivity to other antibiotics was measured by growth on meat infusion agar using ’ Sensidiscs ’ (Baltimore Biological Laboratories), antibiotic discs, and standard plate-diffusion methods. The test organism was sensitive to cephaloridine, 30 .g., chloramphenicol 30 g., kanamycin 5 .g., gentamicin 2 j.g., and nalidixic acid 30 g. (concentration on discs). The organism was resistant to penicillin 10 units, methicillin 5 g., ampicillin 10 g., erythromycin 15 g., streptomycin 10 tg., tetracycline 30 µg., polymyxin B 300 units, colistin 10 µg., nitrofurantoin 100 µg., sulphadiazine 1 mg., sulphafurazole 2 mg. (concentrations on discs). 780 g. per

Culture Media Bacteria were grown in 1% peptone water containing 1 % lactose. Human serum was added to a final concentration of 10% and the medium was boiled for 3 minutes in a water bath (see Results). The inoculum was suspended in 1 % peptone water. Experiments were carried out during incubation in a shaking water-bath controlled to 37(±0.1)°C. Chemical Methods Lactose was measured after hydrolysis and reaction with galactose oxidase and 1% diphenylamine, absorption being read at 540 mµ and compared with standards made from known lactose concentrations in the presence of an inoculum of killed bacteria. pH was measured with a PM 26 meter (Radiometer), measuring to 0.01 pH unit (10 mV per pH), equipped with combined microelectrodes

over 1’/2 hours of pH in a heavily inoculated culture of K. aerogenes in 1% lactose peptone water and 15% boiled pooled serum; no antibiotics.

Fig. 1-Continuous tracing

in each bottle continuously and recorded directly. A typical rate of fall in pH in cultures without antibiotic is shown in fig. 1. Inhibition by Antibiotic In a typical experiment, tubes containing different concentrations of kanamycin from 0 to 250 µg. per ml. dissolved in pooled human serum at a final serum concentration of 10% were set up in triplicate and inoculated with a K. aerogenes suspension. After incubation for 1½ hours in a shaking water-bath at 37°C the tubes were transferred to 0°C in an ice water-bath and the pH was measured at that temperature. Fig. 2 indicates the doseresponse nature of the inhibition of acid production, and

(Titron, Melbourne, Australia, catalogue no. 131 AR). For use with the second method to be described, they were connected through a bridge-circuit buffer adjustment for individual electrodes

and

switched for

discontinuous

recording from any of four electrodes measuring simultaneously. A’Speedomex H ’(Leeds and Northrup) recorder with adjustable zero and range was used at a chart speed of 6 in. (15 cm.) per hour. Serum from Patients Sera were obtained from various hospitals by courtesy of the clinical microbiologists. Dr. S. Bell (Prince of Wales Hospital, Sydney, N.S.W.) kindly provided most of the sera, on which he had carried out plate assays. Results pH without Antibiotic Bottles containing 4 ml. of

Fall in

1% peptone with 1-43,,lactose and 1 ml. pooled human serum were boiled for 3 minutes as described, and inoculated with 1 ml. K. aerogenes suspension. pH was measured water

Fig. 2-Final pH observed after li/z hours in parallel cultures of K. aerogenes in lactose/peptone-water and 10% serum, containing different concentrations of kanamycin. Starting pH 7-8. • =observed point. x =average of triplicates.

377

the

of variability to be expected between tripliThe initial pH in each series was 7-8: the maximum fall in pH after P/2 hours’ incubation was 1-2 pH units with a kanamycin concentration of 1 g. per ml. Antibiotic concentration in patients’ sera could be read from the final pH achieved and referred to the standard curve obtained with known concentrations of kanamycin. amount

cates.

pH

CORRESPONDING COMPARED CONCENTRATIONS MEASURED BY PLATE ASSAY

TABLE

II-FALL

CENTRATIONS

IN

IN

AND

PATIENTS’

SERA

KANAMYCIN WITH

CONKANAMYCIN

Interfering Factors When sera from different patients were first tested, results were widely and unsystematically divergent from

kanamycin content estimated by plate assay. Possible factors causing these disagreements could be individual differences between sera in their antibacterial activity, concentration of glucose or urea, binding of antibiotic, buffering capacity and initial pH, and presence of antibiotics other than the one tested. Heating to 56°C for 30 minutes to destroy antibacterial activity did not alter the discrepancy. There was no change in the final pH when urea or glucose were added to serum samples in concentrations equivalent to serumlevels of 150 and 300 mg. per 100 ml., respectively. Serum binding was not investigated in detail. TABLE I-EFFECT OF INITIAL pH AND BUFFERING OF THE CULTURE MEDIUM ON THE AMOUNT OF ACID PRODUCED AND THE UPTAKE OF LACTOSE BY K. AEROGENES

However, initial pH and buffering were important. sets of twelve tubes, in triplicate, were set up

Three

0-2 ml. of 0-01M phosphate, or phosphatecitrate buffer of initial pH 7-54, 6-92, or 5-90. A fourth series of tubes contained no buffer. To each tube was added 0-2 ml. pooled human serum, 1-4 ml. lactose broth (1-43% to give a final 1% concentration), and 0-2 ml. of the inoculum. All tubes were incubated for 11/2 hours in a shaking water-bath. pH was measured in all tubes. In one series of tubes, lactose concentration was measured; in another series acid was titrated directly with 0° 1N sodium hydroxide and 0-01N hydrochloric acid, using the pH meter to measure the end-point. The results are shown in table i, from which it is clear that the initial pH at which the serum, and consequently the culture system is set, will influence the amount of lactose utilised, and thus the amount of acid produced; and that more acid is produced in buffered medium than in unbuffered medium

containing

the same starting pH. The pH of medium containing sera from different patients varied as much as 0-2 units. Consequently, in order to remove the effect of individual differences in initial pH and in buffering capacity, specimens of serum

at

* This

patient’s serum also contained chloramphenicol organism was sensitive.

were

to

which the

test

diluted 1 in 8 with peptone water, boiled in a waterapproximately 3 minutes, and cooled before

bath for

the test system. The rationale was that boiling would destroy individual differences in serumbuffering capacity by removing carbon dioxide and by denaturing the serum-proteins. Table II shows results obtained after boiling, compared with the results of plate assays. It is clear that there was good agreement. Among other antibiotics to which the test organism was sensitive which are likely to be present in patients’ sera, cephaloridine would be destroyed by boiling, and nalidixic acid is very rapidly excreted in the urine after administration (Carroll 1963). Thus, the presence of chloramphenicol and gentamicin in serum would be likely to influence the test using this organism (the organism was insensitive to eleven other antibiotics included in the sensidiscs). Experimental Errors and Technical Points Errors will depend on the accuracy of pH measurement; the reproducibility of replicate tests; and the shape and accuracy of standard curves, which depend in turn on the accuracy with which the standard solutions are made up and where on the curve the test serum concentration lies, because kanamycin concentration is plotted logarithmically. The approximate error of estimation observed was of the order of ±4 g. per ml. on the flat part of the standard curve (less than 6 jjLg. per ml. kanamycin in serum concentration), and approximately 1 g. per ml. on ttle linear slope (10-100 g. per ml.). The inoculum was grown on medium containing lactose to induce enzyme activity and thereby reduce lag in fermentation during the test. Shaking was essential for smooth curves of pH changes. Temperature control was also maintained within close limits to facilitate reproducibility of results. The deposition of a protein film led to clogging of the glass electrode after some months. The film could be removed by soaking the electrode in protease solution.

adding

the

to

serum

Rate

of pH Change

measuring the final pH after 1112 hours’ incubation, we measured the rate of change of pH continuously during incubation, thus achieving a measure of Instead of

antibiotic concentration within about 40 minutes. A multichannel recorder or a single-channel recorder which measures output discontinuously but serially from several electrodes was used. The result was a slope of change of pH versus time, corresponding to kanamycin concentration when the slope was maximum. (This slope ApH/time is sigmoid in shape. It reaches a maximum value and is

378 K. aerogenes on a nutrient-agar plate; the bacteria are removed with a loop and suspended in 20 ml. of peptone water. The inoculum of 0-20 ml. of this suspension gives a bacterial suspension of 109 to 4 x 109 organisms per ml. in the tubes. All tubes are then incubated at 37 °C in a shaking waterbath with vigorous agitation for 11/2 hours. The tubes are then cooled to 0°C and the pH measured, using a glass combined microelectrode and a suitable pH meter, to the nearest 0-01 pH unit. Draw a standard curve of change in pH (the difference between control and test tubes) and logarithm of kanamycin concentration. The change in pH is calculated for each of the sera and the kanamycin concentration read from the graph. Method 2

Rate of pH change is measured under " rate of pH change ".

as

described in Results

Discussion These two methods involve a new principle in the application of microbiological assays to routine laboratory Fig. 3-Composite from recorder tracings of rate of change of pH in assays containing 0, 10, 20, and 30 fg. per mI. kanamycin (method 2).

virtually linear, for suspensions containing no antibiotic, at about 40 minutes. At this time, the slopes of all suspensions may be measured as tan 9, where 6 is the angle between the slope of the curve and the vertical. Tan 6 is then plotted against log antibiotic concentration for known standards. Unknown values may then be read by measuring tan e and reading antibiotic concentration from the standard curve.) The error of estimation was about ±5% in measuring the slope, corresponding to about ±0-5 .g. per ml. for blood-levels from 0

to

10 g. per

ml., and ±1-3 g. per ml. in the range 20-50 g. per ml. Fig. 3 is reproduced from typical recordings for four different concentrations of kanamycin. Essentially similar results and a similar dose-response standard curve have been obtained using gentamicin in place of kanamycin. Method for Practical Use Method1

Three tubes are required for each specimen, and twenty-four for standards. Serological (13 x 100 mm.) tubes with caps are satisfactory. Distribute 0-70 ml. culture medium (1-43% lactose, 1% peptone water) into each and sterilise by autoclaving at 10 lb. per sq. in. for 10 minutes. Add 0-10 ml. volumes of serum to each tube as follows: into the standard tubes measure sterile pooled human serum containing known concentrations of dissolved kanamycin, using 11 duplicates (twenty-two tubes) with kanamycin concentrations in the range of 0-100 {g. per ml. The remaining two standard tubes are controls containing Measure 0-10 ml. of the serum without antibiotic. patient’s serum into each of the three specimen tubes. All tubes are then heated to 90 °C for 3 minutes, allowing time for the heat to penetrate the medium, in a water-bath. The tubes are cooled to 0°C and inoculated at that temperature. The two standard control tubes and one of each of the triplicate test tubes receive an 0-20 ml. of inoculum prepared as below, but which is heat-killed at 90°C for 10 minutes. The remaining tubes each receive 0-20 ml. of a live inoculum. The inoculum is prepared by growing a lawn of

diagnostic methods. They involve the instrumental of changes taking place rapidly, consequent upon the metabolic activities of bacteria heavily inoculated

measurement

into

a

suitable culture medium.

The

most

obvious

advantages of the application of this principle are time and accuracy. Using the methods described here, blood-levels of antibiotic may be obtained in from 40 to 90 minutes, at least as accurately as by plate-diffusion methods. Tests may be carried out on small volumes of serum which make the methods ideally suited for use in paediatric practice. Although the new methods require different skills and equipment from those usually used in microbiological laboratories, they are practicable, because nothing very complex is required. The slightly more elaborate equipment is not generally used in microbiology diagnostic work, but is nonetheless available in most major laboratories. Several tests may be carried out at the one time by using several electrodes connected through a suitable switch mechanism into a multichannel recording system. The methods are applicable to cerebrospinal fluid, urine, sputum, or any other body fluids. An important practical point is that the inoculum needs only a very simple preparation and can be kept in the cold for at least all of each working-day to allow a single preparation once daily in a routine laboratory. Standard curves have been very reproducible, but should be

repeated daily. The initial expense involves the acquisition of pH meter, electrodes, and recorder, although the recorder is not absolutely necessary for the first method. The cost of such instruments is of the order of a total of$A2000 in Australia. Against this must be balanced the saving in technician time, in preparation of special assay media and plates, setting up multiple dilutions, and prolonged time reading of results. The advantage of rapid information to physicians cannot be calculated in financial terms. The output from the recorders and meters can be connected to integrated laboratory-data-processing systems. We thank Bristol Laboratories Pty. Ltd. for financial help and for supplying the kanamycin, Schering (Australia) Ltd. for a gift of crystalline gentamicin, and Dr. D. C. Dorman, Dr. S. Bell, and Mr. A. Perceval for supplying cultures and sera and advice. Requests for reprints should be addressed to S. F. REFERENCES

Carroll, G. (1963) J. Urol. 90, 476. Miles, A. A., Misra, S. S. (1938) J. Hyg., Camb. 38, 732.